Assessment of a Potential Role of Dickeya dadantii DSM 18020 as a Pectinase Producer for Utilization in Poultry Diets Based on in silico Analyses.

Currently, the poultry industry has been faced with consumer pressure to utilize only vegetable feedstuffs in poultry diets, eliminate antibiotics from poultry production, and rear poultry in free range systems. To maintain current production standards, the industry must determine ways to enhance nutrient uptake and utilization further. One possible solution is the supplementation of pectinase, an enzyme that degrades pectin within the cell walls of plants, in poultry diets. Therefore, the objective of the current study was to determine the potential role of a pectinase producer, Dickeya dadantii DSM 18020, as a commercially utilized pectinase producer in poultry diets against other known pectinase producers, in silico. In the current study, whole genomes of Dickeya dadantii DSM 18020 (Dd18020), D. dadantii 3937 (Dd3937), D. solani IPO 2222 (Ds2222), Bacillus halodurans C-125 (BhC125), and B. subtilis subsp. subtilis str. 168 (Bs168) were compared using bioinformatic approaches to compare the chromosomal genome size, GC content, protein coding genes (CDS), total genes, average protein length (a.a.) and determine the predicted metabolic pathways, predicted pectin degrading enzymes, and pectin-degradation pathways across pectinase producers. Due to insufficient information surrounding the genome of Dd18020 (lack of annotation), the genome of Dd3937, a 99% identical genome to Dd18020, was utilized to compare pectinase-associated enzymes and pathways. The results from the current study demonstrated that Dd3937 possessed the most significant proportion of pathways presented and the highest number of pathways related to degradation, assimilation, and utilization of pectin. Also, Dd18020 exhibited a high number of pectinase-related enzymes. Both Dd3937 and Dd2222 shared the pectin degradation I pathway via the EC 3.1.1.11, EC 3.2.1.82, and EC 4.2.2.- enzymes, but did not share this pathway with either Bacillus species. In conclusion, Dd18020 demonstrated the genetic potential to produce multiple pectinase enzymes that could be beneficial to the degradation of pectin in poultry diets. However, for Dd18020 to become a commercially viable enzyme producer for the poultry industry, further research quantifying the pectinase production in vitro and determining the stability of the produced pectinases during feed manufacturing are necessary.

Draft Genome Sequence of Anoxybacillus sp. Strain UARK-01, a New Thermophilic Lignin-Utilizing Bacterium Isolated from Soil in Arkansas, USA.

The draft genome of Anoxybacillus sp. strain UARK-01, a novel lignin-utilizing thermophilic soil bacterium, represents the first sequence of an Anoxybacillus isolate from the United States. The genome was sequenced using the Illumina MiSeq platform, de novo assembled using SeqMan NGen, and annotated at NCBI. The genome sequence revealed genes for laccase and lignocellulose degradation enzymes.

Anoxybacillus sp. Strain UARK-01, a New Thermophilic Soil Bacterium with Hyperthermostable Alkaline Laccase Activity.

We describe the isolation and characteristics of a novel thermophilic bacterium from soil. The organism is a member of the Anoxybacillus genus based on phylogenetic analysis of the 16S rRNA gene. The 16S rRNA of the organism shares >99% sequence identity with those of two species, Anoxybacillus rupiensis and A. geothermalis. We named this isolate as Anoxybacillus sp. strain UARK-01. UARK-01 grows optimally in the presence of oxygen at 55 °C and pH 8. It grew excellently in the presence of lignin as the sole carbon source. Culture supernatant from UARK-01 grown on lignin was rich in laccase activity. The laccase activity was optimal at 90 °C and pH 9, and there was comparable activity at 80 and 100 °C. The crude laccase decolorized approximately 75% of Congo Red in 7 h under optimal conditions. A single laccase gene was identified from the draft genome sequence of Anoxybacillus sp. UARK-01. The UARK-01 laccase (Anox_Lacc) was cloned and overexpressed in Escherichia coli and was partially purified. The partially purified Anox_Lacc decolorized approximately 1.64+/0.21 nanomoles of Congo Red per microgram protein in 30 min at 90 °C and pH 9. Anox_Lacc is a member of the multicopper polyphenol oxidoreductase laccase family (pfam02578 Cu-oxidase_4) and has novel characteristics. Multiple sequence analysis of Anox_Lacc with six homologs from the family revealed four conserved copper ligands and several new residues that are fully conserved. Anox_Lacc is enriched in leucine, glutamine, and lysine, and it contains fewer alanine, arginine, glycine, and serine residues. Skewed amino acid composition of Anox_Lacc likely contributes to the exceptional thermochemical properties of the laccase activity from UARK-01. Both lignin utilization and production of hyperthermostable alkaline laccase are new findings in the Anoxybacillus genus.

Complete genome sequence of Arthrobacter sp. strain FB24.

Arthrobacter sp. strain FB24 is a species in the genus Arthrobacter Conn and Dimmick 1947, in the family Micrococcaceae and class Actinobacteria. A number of Arthrobacter genome sequences have been completed because of their important role in soil, especially bioremediation. This isolate is of special interest because it is tolerant to multiple metals and it is extremely resistant to elevated concentrations of chromate. The genome consists of a 4,698,945 bp circular chromosome and three plasmids (96,488, 115,507, and 159,536 bp, a total of 5,070,478 bp), coding 4,536 proteins of which 1,257 are without known function. This genome was sequenced as part of the DOE Joint Genome Institute Program.

Effects of phenolic monomers on growth of Acidothermus cellulolyticus.

Previous studies on biological pretreatment of switchgrass by solid-state fermentation with Acidothermus cellulolyticus 11B have shown that inhibitory compounds prevent growth on untreated switchgrass. A. cellulolyticus was grown in liquid medium containing cellobiose with phenolic monomers added to determine if the phenolic compounds are one possible source of inhibition. Cinnamic acid derivatives (trans-p-coumaric, trans-ferulic, and hydrocinnamic acids), hydroxybenzoic acids (p-hydroxybenzoic, syringic, and vanillic acids), benzaldehydes (vanillin and p-hydroxybenzaldehyde), and condensed tannin monomers (catechin and epicatechin) were tested at levels up to 20 mM. All compounds exhibited a dose-response relationship and strongly inhibited growth at 20 mM. trans-p-Coumaric acid was found to be the strongest inhibitor of A. cellulolyticus growth, with a specific growth rate of 0.004 h(-1) at 1 mM (0.18 h(-1) without phenolic monomer). GC-MS and HPLC methods were used to confirm the presence of these phenolic compounds in switchgrass and measure the amounts extracted using different conditions. The amounts of phenolic compounds measured were found to be higher than the threshold for growth inhibition. Leaching with water at 55°C was inefficient at removing bound phenolics, whereas NaOH treatment improved efficiency. Phenolic compounds spiked into alkaline pretreated switchgrass were also found to inhibit growth of A. cellulolyticus in solid-state fermentation. However, addition of insoluble polyvinylpolypyrrolidone (PVPP) to switchgrass improved growth of A. cellulolyticus in liquid cultures, providing a possible approach for alleviating microbial inhibition due to phenolic compounds in lignocellulose.

New perspectives on nodule nitrogen assimilation in actinorhizal symbioses.

Nitrogen-fixing root nodules are plant organs specialised for symbiotic transfer of nitrogen and carbon between microsymbiont and host. The organisation of nitrogen assimilation, storage and transport processes is partitioned at the subcellular and tissue levels, in distinctive patterns depending on the symbiotic partners. In this review, recent advances in understanding of actinorhizal nodule nitrogen assimilation are presented. New findings indicate that Frankia within nodules of Datisca glomerata (Presl.) Baill. carries out both primary nitrogen assimilation and biosynthesis of arginine, rather than exporting ammonium. Arginine is a typical storage form of nitrogen in plant tissues, but is a novel nitrogen carrier molecule in root nodule symbioses. Thus Frankia within D. glomerata nodules exhibits considerable metabolic independence. Furthermore, nitrogen reassimilation is likely to take place in the host in the uninfected nodule cortical cells of this root nodule symbiosis, before amino acid export to host sink tissues via the xylem. The role of an augmented pericycle in carbon and nitrogen exchange in root nodules deserves further attention in actinorhizal symbiosis, and further highlights the importance of a comprehensive, structure-function approach to understanding function in root nodules. Moreover, the multiple patterns of compartmentalisation in relation to nitrogen flux within root nodules demonstrate the diversity of possible functional interactions between host and microsymbiont that have evolved in the nitrogen-fixing clade.

Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus 11B.

We cloned and purified the major family 10 xylanase (Xyn10A) from Acidothermus cellulolyticus 11B. Xyn10A was active on oat spelt and birchwood xylans between 60°C and 100°C and between pH 4 and pH 8. The optimal activity was at 90°C and pH 6; specific activity and K(m) for oat spelt xylan were 350 µmol xylose produced min⁻¹ mg of protein⁻¹ and 0.53 mg ml⁻¹, respectively. Based on xylan cleavage patterns, Xyn10A is an endoxylanase, and its half-life at 90°C was approximately 1.5 h in the presence of xylan.

Large direct repeats flank genomic rearrangements between a new clinical isolate of Francisella tularensis subsp. tularensis A1 and Schu S4.

Francisella tularensis subspecies tularensis consists of two separate populations A1 and A2. This report describes the complete genome sequence of NE061598, an F. tularensis subspecies tularensis A1 isolated in 1998 from a human with clinical disease in Nebraska, United States of America. The genome sequence was compared to Schu S4, an F. tularensis subspecies tularensis A1a strain originally isolated in Ohio in 1941. It was determined that there were 25 nucleotide polymorphisms (22 SNPs and 3 indels) between Schu S4 and NE061598; two of these polymorphisms were in potential virulence loci. Pulsed-field gel electrophoresis analysis demonstrated that NE061598 was an A1a genotype. Other differences included repeat sequences (n = 11 separate loci), four of which were contained in coding sequences, and an inversion and rearrangement probably mediated by insertion sequences and the previously identified direct repeats I, II, and III. Five new variable-number tandem repeats were identified; three of these five were unique in NE061598 compared to Schu S4. Importantly, there was no gene loss or gain identified between NE061598 and Schu S4. Interpretation of these data suggests there is significant sequence conservation and chromosomal synteny within the A1 population. Further studies are needed to determine the biological properties driving the selective pressure that maintains the chromosomal structure of this monomorphic pathogen.

Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00.

Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD(23) deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997-1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also approximately 5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1-1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H(+) antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.

Comprehensive analyses of transport proteins encoded within the genome of "Aromatoleum aromaticum" strain EbN1.

The denitrifying bacterium "Aromatoleum aromaticum" strain EbN1 is specialized for the aerobic utilization of aromatic compounds including crude oil constituents. We here report whole-genome analyses for potential transport proteins in A. aromaticum strain EbN1. This organism encodes very few transporters for simple sugars and most other common carbon sources. However, up to 28% of its putative transporters may act on fairly hydrophobic aromatic and aliphatic compounds. We categorize the putative transporters encoded within the genome, assign them to recognized families, and propose their preferred substrates. The bioinformatic data are correlated with available metabolic information to obtain an integrated view of the metabolic network of A. aromaticum strain EbN1. The results thus indicate that this organism possesses a disproportionately large percentage of transporters for the uptake and efflux of hydrophobic and amphipathic aromatic and aliphatic compounds compared with previously analyzed organisms. We predict that these findings will have important implications for our ecophysiological understanding of bioremediation.

Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations.

We present here the complete 2.4-Mb genome of the cellulolytic actinobacterial thermophile Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized and elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls, and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. Several of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymes are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and noncoding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot-springs-dwelling prokaryote include a low occurrence of pseudogenes or mobile genetic elements, an unexpected complement of flagellar genes, and the presence of three laterally acquired genomic islands of likely ecophysiological value.

Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils.

The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N(2) fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum).

This paper describes the genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the Wood-Ljungdahl pathway of CO and CO(2) fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using H(2) and CO as electron donors and CO(2) as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO(2). Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols and aromatic compounds) and electron acceptors (CO(2), nitrate, nitrite, thiosulfate, dimethylsulfoxide and aromatic carboxyl groups). The genome consists of a single circular 2 628 784 bp chromosome encoding 2615 open reading frames (ORFs), which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. A total of 2384 (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in orthologue clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).

Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.

Comprehensive analysis of transport proteins encoded within the genome of Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the Transporter Classification Database. A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least four types of inner-membrane secretion systems and five types of outer-membrane secretion systems are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer-membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer-membrane transport and motility than does Escherichia coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes are also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses.

Transport capabilities of eleven gram-positive bacteria: comparative genomic analyses.

The genomes of eleven Gram-positive bacteria that are important for human health and the food industry, nine low G+C lactic acid bacteria and two high G+C Gram-positive organisms, were analyzed for their complement of genes encoding transport proteins. Thirteen to 18% of their genes encode transport proteins, larger percentages than observed for most other bacteria. All of these bacteria possess channel proteins, some of which probably function to relieve osmotic stress. Amino acid uptake systems predominate over sugar and peptide cation symporters, and of the sugar uptake porters, those specific for oligosaccharides and glycosides often outnumber those for free sugars. About 10% of the total transport proteins are constituents of putative multidrug efflux pumps with Major Facilitator Superfamily (MFS)-type pumps (55%) being more prevalent than ATP-binding cassette (ABC)-type pumps (33%), which, however, usually greatly outnumber all other types. An exception to this generalization is Streptococcus thermophilus with 54% of its drug efflux pumps belonging to the ABC superfamily and 23% belonging each to the Multidrug/Oligosaccharide/Polysaccharide (MOP) superfamily and the MFS. These bacteria also display peptide efflux pumps that may function in intercellular signalling, and macromolecular efflux pumps, many of predictable specificities. Most of the bacteria analyzed have no pmf-coupled or transmembrane flow electron carriers. The one exception is Brevibacterium linens, which in addition to these carriers, also has transporters of several families not represented in the other ten bacteria examined. Comparisons with the genomes of organisms from other bacterial kingdoms revealed that lactic acid bacteria possess distinctive proportions of recognized transporter types (e.g., more porters specific for glycosides than reducing sugars). Some homologues of transporters identified had previously been identified only in Gram-negative bacteria or in eukaryotes. Our studies reveal unique characteristics of the lactic acid bacteria such as the universal presence of genes encoding mechanosensitive channels, competence systems and large numbers of sugar transporters of the phosphotransferase system. The analyses lead to important physiological predictions regarding the preferred signalling and metabolic activities of these industrially important bacteria.

Efflux pump gene expression in Erwinia chrysanthemi is induced by exposure to phenolic acids.

Salicylic acid (SA) is an important signaling molecule in local and systemic plant resistance. Following infection by microbial pathogens and the initial oxidative burst in plants, SA accumulation functions in the amplification of defense gene expression. Production of pathogenesis-related proteins and toxic antimicrobial chemicals serves to protect the plant from infection. Successful microbial pathogens utilize a variety of mechanisms to rid themselves of toxic antimicrobial compounds. Important among these mechanisms are multidrug-resistance pumps that bring about the active efflux of toxic compounds from microbial cells. Here, we show that a combination SA and its precursors, t-cinnamic acid and benzoic acid, can activate expression of specific multidrug efflux pump-encoding genes in the plant pathogen Erwinia chrysanthemi and enhance survival of the bacterium in the presence of model as well as plant-derived antimicrobial chemicals. This ability of plant-pathogenic bacteria to co-opt plant defense-signaling molecules to activate multidrug efflux pumps may have evolved to ensure bacterial survival in susceptible host plants.

Topological predictions for integral membrane permeases of the phosphoenolpyruvate:sugar phosphotransferase system.

We report bioinformatic analyses of the largest superfamily of integral membrane permeases of the bacterial phosphotransferase system (PTS), the Enzyme IIC constituents of the Glc superfamily. Phylogenetic analyses reveal that this superfamily consists of five equally distant families, the Glucose (Glc), beta-Glucoside (Bgl), Fructose (Fru), Mannitol (Mtl) and Lactose (Lac) families. Average hydropathy, amphipathicity and similarity plots were generated for these five families as well as for the entire superfamily. Charged residue distribution was analyzed, and the most conserved sequence motif, common to all five families, was identified. The results show that the members of all five families exhibit similar average hydropathy plots with regions of average amphipathicity and relative conservation also being similar. Evidence is presented suggesting that the Glucitol (Gut) family of Enzyme IIC constituents is a distant member of the Glc superfamily. Based on our analyses we offer a topological model that resembles, but differs in detail from the two previously proposed models.

Extra domains in secondary transport carriers and channel proteins.

"Extra" domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA(Fru) and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane alpha-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.