DNA Conformation Induces Adaptable Binding by Tandem Zinc Finger Proteins.

Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution.

A Milieu Molecule for TGF-ß Required for Microglia Function in the Nervous System.

Extracellular proTGF-ß is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-ß is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVß8-dependent TGF-ß activation. Lrrc33-/- mice lack CNS vascular abnormalities associated with deficiency in TGF-ß-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33-/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33-/- microglia in the same brain suggest that there is little spreading of TGF-ß activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-ß provide localized and selective activation of TGF-ß.

Two broadly conserved families of polyprenyl-phosphate transporters.

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

SYNBIP: synthetic binding proteins for research, diagnosis and therapy.

The success of protein engineering and design has extensively expanded the protein space, which presents a promising strategy for creating next-generation proteins of diverse functions. Among these proteins, the synthetic binding proteins (SBPs) are smaller, more stable, less immunogenic, and better of tissue penetration than others, which make the SBP-related data attracting extensive interest from worldwide scientists. However, no database has been developed to systematically provide the valuable information of SBPs yet. In this study, a database named 'Synthetic Binding Proteins for Research, Diagnosis, and Therapy (SYNBIP)' was thus introduced. This database is unique in (a) comprehensively describing thousands of SBPs from the perspectives of scaffolds, biophysical & functional properties, etc.; (b) panoramically illustrating the binding targets & the broad application of each SBP and (c) enabling a similarity search against the sequences of all SBPs and their binding targets. Since SBP is a human-made protein that has not been found in nature, the discovery of novel SBPs relied heavily on experimental protein engineering and could be greatly facilitated by in-silico studies (such as AI and computational modeling). Thus, the data provided in SYNBIP could lay a solid foundation for the future development of novel SBPs. The SYNBIP is accessible without login requirement at both official (https://idrblab.org/synbip/) and mirror (http://synbip.idrblab.net/) sites.

Genome-wide identification and characterization of nonspecific lipid transfer protein (nsLTP) genes in Arachis duranensis.

Nonspecific lipid transfer proteins (nsLTPs) play vital roles in lipid metabolism, cell apoptosis and biotic and abiotic stresses in plants. However, the distribution of nsLTPs in Arachis duranensis has not been fully characterized. In this study, we identified 64 nsLTP genes in A. duranensis (designated AdLTPs), which were classified into six subfamilies and randomly distributed along nine chromosomes. Tandem and segmental duplication events were detected in the evolution of AdLTPs. The Ks and ω values differed significantly between Types 1 and D subfamilies, and eight AdLTPs were under positive selection. The expression levels of AdLTPs were changed after salinity, PEG, low-temperature and ABA treatments. Three AdLTPs were associated with resistance to nematode infection, and DOF and WRI1 transcription factors may regulate the AdLTP response to nematode infection. Our results may provide valuable genomic information for the breeding of peanut cultivars that are resistant to biotic and abiotic stresses.

The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region.

The homeodomain-interacting protein kinase (HIPK) family is comprised of four nuclear protein kinases, HIPK1-4. HIPK proteins phosphorylate a diverse range of transcription factors involved in cell proliferation, differentiation, and apoptosis. HIPK2, thus far the best-characterized member of this largely understudied family of protein kinases, plays a role in the activation of p53 in response to DNA damage. Despite this tumor-suppressor function, HIPK2 is also found overexpressed in several cancers, and its hyperactivation causes chronic fibrosis. There are currently no structures of HIPK2 or of any other HIPK kinase. Here, we report the crystal structure of HIPK2's kinase domain bound to CX-4945, a casein kinase 2α (CK2α) inhibitor currently in clinical trials against several cancers. The structure, determined at 2.2 Å resolution, revealed that CX-4945 engages the HIPK2 active site in a hybrid binding mode between that seen in structures of CK2α and Pim1 kinases. The HIPK2 kinase domain crystallized in the active conformation, which was stabilized by phosphorylation of the activation loop. We noted that the overall kinase domain fold of HIPK2 closely resembles that of evolutionarily related dual-specificity tyrosine-regulated kinases (DYRKs). Most significant structural differences between HIPK2 and DYRKs included an absence of the regulatory N-terminal domain and a unique conformation of the CMGC-insert region and of a newly defined insert segment in the αC-ß4 loop. This first crystal structure of HIPK2 paves the way for characterizing the understudied members of the HIPK family and for developing HIPK2-directed therapies for managing cancer and fibrosis.

Molecular characterization and expression patterns of two LPS binding /bactericidal permeability-increasing proteins (LBP/BPIs) from the scallop Argopecten purpuratus.

Lipopolysaccharide-binding proteins (LBPs) and bactericidal permeability-increasing proteins (BPIs) are effectors of the innate immune response which act in a coordinated manner to bind and neutralize the LPS present in Gram negative bacteria. The structural organization that confers the function of LBPs and BPIs is very similar, however, they are antagonistic to each other. In this work, we characterized two LBP/BPIs from the scallop Argopecten purpuratus, namely ApLBP/BPI1 and ApLBP/BPI2. The molecular and phylogenetic analyses of ApLBP/BPIs indicated that both isoforms display classic characteristics of LBP/BPIs from other invertebrates. Additionally, ApLBP/BPIs are constitutively expressed in scallop tissues and their transcript expression is upregulated in hemocytes and gills in response to an immune challenge. However, some structural characteristics of functional importance for the biological activity of these molecules, such as the net charge differ substantially between ApLBP/BPI1 and ApLBP/BPI2. Furthermore, each isoform displays a specific profile of basal expression among different tissues, as well as specific patterns of expression during the activation of the immune response. Results suggest that functional specialization of ApLBP/BPIs might happen, with potential role as LBP or BPI in this species of scallop. Further research on the biological activities of ApLBP/BPIs are necessary to elucidate their participation in the scallop immune response.

Endosomal sorting and signalling: an emerging role for sorting nexins.

The endocytic network comprises a series of interconnected tubulo-vesicular membranous compartments that together regulate various sorting and signalling events. Although it is clear that defects in endocytic function underlie a variety of human diseases, our understanding of the molecular entities that regulate these sorting and signalling events remains limited. Here we discuss the sorting nexins family of proteins and propose that they have a fundamental role in orchestrating the formation of protein complexes that are involved in endosomal sorting and signalling.

Structural and Functional Analysis of PGRP-LC Indicates Exclusive Dap-Type PGN Binding in Bumblebees.

Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Bees possess fewer PGRPs than Drosophila melanogaster and Anopheles gambiae but retain two important immune pathways, the Toll pathway and the Imd pathway, which can be triggered by the recognition of Dap-type PGN by PGRP-LCx with the assistance of PGRP-LCa in Drosophila. There are three isoforms of PGRP-LC including PGRP-LCx, PGRP-LCa and PGRP-LCy in Drosophila. Our previous study showed that a single PGRP-LC exists in bumblebees. In this present study, we prove that the bumblebee Bombus lantschouensis PGRP-LC (Bl-PGRP-LC) can respond to an infection with Gram-negative bacterium Escherichia coli through binding to the Dap-type PGNs directly, and that E. coli infection induces the quick and strong upregulation of PGRP-LC, abaecin and defensin. Moreover, the Bl-PGRP-LC exhibits a very strong affinity for the Dap-type PGN, much stronger than the affinity exhibited by the PGRP-LC from the more eusocial honeybee Apis mellifera (Am-PGRP-LC). In addition, mutagenesis experiments showed that the residue His390 is the anchor residue for the binding to the Dap-type PGN and forms a hydrogen bond with MurNAc rather than meso-Dap, which interacts with the anchor residue Arg413 of PGRP-LCx in Drosophila. Therefore, bumblebee PGRP-LC possesses exclusive characteristics for the immune response among insect PGRPs.

Enrichment of ATP Binding Proteins Unveils Proteomic Alterations in Human Macrophage Cell Death, Inflammatory Response, and Protein Synthesis after Interaction with Candida albicans.

Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C. albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C. albicans.

Prominent Binding of Human and Equine Fibrinogen to Streptococcus equi subsp. zooepidemicus Is Mediated by Specific SzM Types and Is a Distinct Phenotype of Zoonotic Isolates.

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.

Comparative genomics of the transportome of Ten Treponema species.

Treponema is a diverse bacterial genus, the species of which can be pathogenic, symbiotic, or free living. These treponemes can cause various diseases in humans and other animals, such as periodontal disease, bovine digital dermatitis and animal skin lesions. However, the most important and well-studied disease of treponemes that affects humans is 'syphilis'. This disease is caused by Treponema pallidum subspecie pallidum with 11-12 million new cases around the globe on an annual basis. In this study we analyze the transportome of ten Treponema species, with emphasis on the types of encoded transport proteins and their substrates. Of the ten species examined, two (T. primitia and T. azonutricium) reside as symbionts in the guts of termites; six (T. pallidum, T. paraluiscuniculi, T. pedis, T. denticola, T. putidum and T. brennaborense) are pathogens of either humans or animals, and T. caldarium and T. succinifaciens are avirulent species, the former being thermophilic. All ten species have a repertoire of transport proteins that assists them in residing in their respective ecological niches. For instance, oral pathogens use transport proteins that take up nutrients uniquely present in their ecosystem; they also encode multiple multidrug/macromolecule exporters that protect against antimicrobials and aid in biofilm formation. Proteins of termite gut symbionts convert cellulose into other sugars that can be metabolized by the host. As often observed for pathogens and symbionts, several of these treponemes have reduced genome sizes, and their small genomes correlate with their dependencies on the host. Overall, the transportomes of T. pallidum and other pathogens have a conglomerate of parasitic lifestyle-assisting proteins. For example, a T. pallidum repeat protein (TprK) mediates immune evasion; outer membrane proteins (OMPs) allow nutrient uptake and end product export, and several ABC transporters catalyze sugar uptake, considered pivotal to parasitic lifestyles. Taken together, the results of this study yield new information that may help open new avenues of treponeme research.

SH3Ps-Evolution and Diversity of a Family of Proteins Engaged in Plant Cytokinesis.

SH3P2 (At4g34660), an Arabidopsis thaliana SH3 and Bin/amphiphysin/Rvs (BAR) domain-containing protein, was reported to have a specific role in cell plate assembly, unlike its paralogs SH3P1 (At1g31440) and SH3P3 (At4g18060). SH3P family members were also predicted to interact with formins-evolutionarily conserved actin nucleators that participate in microtubule organization and in membrane-cytoskeleton interactions. To trace the origin of functional specialization of plant SH3Ps, we performed phylogenetic analysis of SH3P sequences from selected plant lineages. SH3Ps are present in charophytes, liverworts, mosses, lycophytes, gymnosperms, and angiosperms, but not in volvocal algae, suggesting association of these proteins with phragmoplast-, but not phycoplast-based cell division. Separation of three SH3P clades, represented by SH3P1, SH3P2, and SH3P3 of A. thaliana, appears to be a seed plant synapomorphy. In the yeast two hybrid system, Arabidopsis SH3P3, but not SH3P2, binds the FH1 and FH2 domains of the formin FH5 (At5g54650), known to participate in cytokinesis, while an opposite binding specificity was found for the dynamin homolog DRP1A (At5g42080), confirming earlier findings. This suggests that the cytokinetic role of SH3P2 is not due to its interaction with FH5. Possible determinants of interaction specificity of SH3P2 and SH3P3 were identified bioinformatically.

Acyl-CoA-binding protein family members in laticifers are possibly involved in lipid and latex metabolism of Hevea brasiliensis (the Para rubber tree).

BACKGROUND: Acyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and their various functions have been characterized in model plants, such as Arabidopsis thaliana (A. thaliana), Oryza sativa (rice), and other plant species. In the present study, genome-wide mining and expression analysis of ACBP genes was performed on Hevea brasiliensis (the para rubber tree), the most important latex-producing crop in the world. RESULTS: Six members of the H. brasiliensis ACBP family genes, designated HbACBP1-HbACBP6, were identified from the H. brasiliensis genome. They can be categorized into four classes with different amino acid sequences and domain structures based on the categorization of their A. thaliana counterparts. Phylogenetic analysis shows that the HbACBPs were clustered with those of other closely related species, such as Manihot esculenta, Ricinus communis, and Jatropha carcas, but were further from those of A. thaliana, a distantly related species. Expression analysis demonstrated that the HbACBP1 and HbACBP2 genes are more prominently expressed in H. brasiliensis latex, and their expression can be significantly enhanced by bark tapping (a mechanical wound) and jasmonic acid stimulation, whereas HbACBP3-HbACBP6 had almost the same expression patterns with relatively high levels in mature leaves and male flowers, but a markedly low abundance in the latex. HbACBP1 and HbACBP2 may have crucial roles in lipid and latex metabolism in laticifers, so their subcellular location was further investigated and the results indicated that HbACBP1 is a cytosol protein, whereas HbACBP2 is an endoplasmic reticulum-associated ACBP. CONCLUSIONS: In this study, the H. brasiliensis ACBP family genes were identified. Phylogenetic analyses of the HbABCPs indicate that there is a high conservation and evolutionary relationship between ACBPs in land plants. The HbACBPs are organ/tissue-specifically expressed and have different expression patterns in response to stimulation by bark tapping or ethrel/jasmonic acid. HbACBP1 and HbACBP2 are two important latex ACBPs that might be involved in the lipid and latex metabolism. The results may provide valuable information for further investigations into the biological functions of HbACBPs during latex metabolism and stress responses in H. brasiliensis.

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M-protein antigens derived from Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N-terminal fragment of M protein, has been developed. Live bacterial-vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10-valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three- to 10-fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10-immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10-immunized mice did not differ significantly from that of unimmunized mice. Mx-10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

Six indicator genes for zinc (Zn) homeostasis in freshwater teleost yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional changes to Zn exposure.

Excessive Zn in the aquatic environment can be toxic and causes dysfunction in Zn homeostasis for fish, which ultimately influences the function of various biological processes. Zn homeostasis is controlled by Zn transporters. This study cloned and characterized the full-length cDNA sequences of six Zn transport-relevant genes (ZnT1, ZnT5, ZnT7, ZIP4, ZIP5 and MTF-1) from yellow catfish Pelteobagrus fulvidraco. The six genes share similar domains to their corresponding members of mammals. Their mRNA amounts were widely existent across eight tissues (intestine, liver, brain, heart, gill, muscle, spleen and mesenteric fat), but relatively predominant in the liver and intestine. On day 28, Zn exposure tended to increase transcript levels of ZnT1, ZnT5 and MTF-1, decrease hepatic ZIP5 expression, but did not significantly affect the expression of ZnT7 and ZIP4. On day 56, Zn exposure tended to increase transcript levels of ZnT1 and MTF-1, down-regulate hepatic mRNA amounts of ZIP4 and ZIP5; among three Zn treatments, ZnT5 expression in the 0.5 mg Zn/L group and ZnT7 expression in the 0.25 mg Zn/L group were the highest. The mRNA abundances of these genes showed Zn concentration- and exposure time-dependent manners. For the first time, we characterized the full-length cDNA sequences of six Zn transport-relevant genes in fish, explored their tissue expression profiles and transcriptional responses to Zn exposure. Our study built good basis for further investigating their physiological functions of these genes and provided new insights into the regulatory mechanisms of Zn homeostasis in fish.

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness.

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a "bottom-up" manner, from their biochemistry and protein-protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.

TRIM-NHL proteins in development and disease.

TRIM-NHL proteins are key regulators of developmental transitions, for example promoting differentiation, while inhibiting cell growth and proliferation, in stem and progenitor cells. Abnormalities in these proteins have been also associated with human diseases, particularly affecting muscular and neuronal functions, making them potential targets for therapeutic intervention. The purpose of this review is to provide a systematic and comprehensive summary on the most studied TRIM-NHL proteins, highlighting examples where connections were established between structural features, molecular functions and biological outcomes.

Expression and putative role of mitochondrial transport proteins in cancer.

Cancer cells undergo major changes in energy and biosynthetic metabolism. One of them is the Warburg effect, in which pyruvate is used for fermentation rather for oxidative phosphorylation. Another major one is their increased reliance on glutamine, which helps to replenish the pool of Krebs cycle metabolites used for other purposes, such as amino acid or lipid biosynthesis. Mitochondria are central to these alterations, as the biochemical pathways linking these processes run through these organelles. Two membranes, an outer and inner membrane, surround mitochondria, the latter being impermeable to most organic compounds. Therefore, a large number of transport proteins are needed to link the biochemical pathways of the cytosol and mitochondrial matrix. Since the transport steps are relatively slow, it is expected that many of these transport steps are altered when cells become cancerous. In this review, changes in expression and regulation of these transport proteins are discussed as well as the role of the transported substrates. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Comparative genomics and evolution of the amylase-binding proteins of oral streptococci.

BACKGROUND: Successful commensal bacteria have evolved to maintain colonization in challenging environments. The oral viridans streptococci are pioneer colonizers of dental plaque biofilm. Some of these bacteria have adapted to life in the oral cavity by binding salivary α-amylase, which hydrolyzes dietary starch, thus providing a source of nutrition. Oral streptococcal species bind α-amylase by expressing a variety of amylase-binding proteins (ABPs). Here we determine the genotypic basis of amylase binding where proteins of diverse size and function share a common phenotype. RESULTS: ABPs were detected in culture supernatants of 27 of 59 strains representing 13 oral Streptococcus species screened using the amylase-ligand binding assay. N-terminal sequences from ABPs of diverse size were obtained from 18 strains representing six oral streptococcal species. Genome sequencing and BLAST searches using N-terminal sequences, protein size, and key words identified the gene associated with each ABP. Among the sequenced ABPs, 14 matched amylase-binding protein A (AbpA), 6 matched amylase-binding protein B (AbpB), and 11 unique ABPs were identified as peptidoglycan-binding, glutamine ABC-type transporter, hypothetical, or choline-binding proteins. Alignment and phylogenetic analyses performed to ascertain evolutionary relationships revealed that ABPs cluster into at least six distinct, unrelated families (AbpA, AbpB, and four novel ABPs) with no phylogenetic evidence that one group evolved from another, and no single ancestral gene found within each group. AbpA-like sequences can be divided into five subgroups based on the N-terminal sequences. Comparative genomics focusing on the abpA gene locus provides evidence of horizontal gene transfer. CONCLUSION: The acquisition of an ABP by oral streptococci provides an interesting example of adaptive evolution.