Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Contribution of Phe-7 to Tat-dependent export of ß-lactamase in Xanthomonas campestris.

Strains of Xanthomonas campestris pv. campestris isolated in Taiwan are commonly resistant to ampicillin owing to the constitutive expression of a chromosomally encoded ß-lactamase that is secreted into the periplasm. In this study, we found that levels of ß-lactamase vary among X. campestris pv. campestris strains, a difference that can be attributed to amino acid substitutions at least at positions 7 and 206, with the former having the major impact. Bioinformatic and PCR analyses indicated that X. campestris pv. campestris possesses tatABC genes and that the signal peptide of X. campestris pv. campestris pre-Bla contains the typical twin-arginine motif (N-R-R-Q-F-L at amino acid residues 3 to 8 in strain X. campestris pv. campestris strain 11), suggesting that Bla is secreted via the Tat pathway. To assess the importance of Phe(7) in the efficient export of X. campestris pv. campestris Bla, we prepared mutant constructs containing amino acid substitutions and monitored their expression by measuring enzyme activity and detecting Bla protein by Western blotting. The results indicate that replacement of Phe(7) with Leu severely inhibited Bla export whereas replacement with Pro almost abolished it. Although a change to Arg caused moderate inhibition of export, replacement with Tyr had no effect. These results suggest that for efficient export of Bla by X. campestris pv. campestris, the aromatic-aromatic interactions and stability of protein structure around the twin-arginine motif are important, since only proteins that can attain a folded state in the cytoplasm are competent for export via the Tat pathway.

Wide host range and strong lytic activity of Staphylococcus aureus lytic phage Stau2.

In searching for an alternative antibacterial agent against multidrug-resistant Staphylococcus aureus, we have isolated and characterized a lytic staphylophage, Stau2. It possesses a double-stranded DNA genome estimated to be about 134.5 kb and a morphology resembling that of members of the family Myoviridae. With an estimated latency period of 25 min and a burst size of 100 PFU/infected cell, propagation of Stau2 in liquid culture gave a lysate of ca. 6 × 10(10) PFU/ml. It was stable at pH 5 to 13 in normal saline at room temperature for at least 4 weeks and at -85°C for more than 2 years, while 1 × 10(9) out of 2 × 10(12) PFU/ml retained infectivity after 36 months at 4°C. Stau2 could lyse 80% of the S. aureus isolates (164/205) obtained from hospitals in Taiwan, with complete lysis of most of the isolates tested within 3 h; however, it was an S. aureus-specific phage because no lytic infection could be found in the coagulase-negative staphylococci tested. Its host range among S. aureus isolates was wider than that of polyvalent phage K (47%), which can also lyse many other staphylococcal species. Experiments with mice demonstrated that Stau2 could provide 100% protection from lethal infection when a multiplicity of infection of 10 was administered immediately after a challenge with S. aureus S23. Considering these results, Stau2 could be considered at least as a candidate for topical phage therapy or an additive in the food industry.

Lytic myophage Abp53 encodes several proteins similar to those encoded by host Acinetobacter baumannii and phage phiKO2.

Acinetobacter baumannii is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant A. baumannii isolates has increased in recent years. Directed toward phage therapy, a lytic phage of A. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to Myoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the A. baumannii isolates tested, which were all multiple drug resistant, but not other bacteria. Mg(2+) enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by A. baumannii strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of Klebsiella phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of A. baumannii ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.

Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity.

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell-cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-beta-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo-beta-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.

Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins.

hspA encodes a small heat shock protein (sHSP) in Xanthomonas campestris pv. campestris, the causative agent of black rot in cruciferous plants. In this study, two-dimensional gel electrophoresis, promoter activity assays, and Northern hybridization results revealed that HspA expression was induced by heat shock but not by other stresses, although low-level expression was detectable by reverse transcription-polymerase chain reaction (RT-PCR) under normal culture conditions. An hspA mutant exhibited reduced tolerance to heat, especially in the presence of MgSO4, but no change in pathogenicity. Results of size-exclusion chromatography indicated that purified HspA(his), containing six C-terminal histidine residues, formed two different size classes of oligomeric complexes--410 and 820 kDa. In contrast, HspA(ter), the unmodified protein translated from the original hspA gene, formed only the 820-kDa complex. These results suggest that the C-terminus of HspA is important for oligomerization. Both HspA820(his) and HspA410(his) were able to partially protect luciferase against heat-induced aggregation. Unlike other reported sHSPs that commonly capture denaturing proteins in refoldable states until refolded by adenosine triphosphate-dependent chaperone systems, HspA(his) alone was capable of reactivating heat-inactivated EcoRI. Thus, Xanthomonas campestris pv. campestris HspA has potential application as a protective agent during the storage of proteins.

Antimicrobial and anti-inflammatory potential of Angelica dahurica and Rheum officinale extract accelerates wound healing in Staphylococcus aureus-infected wounds.

Wound infection is a serious clinical problem, and the most common infection-causing bacteria are Staphylococcus aureus and Pseudomonas aeruginosa. Angelica dahurica and Rheum officinale extract (ARE) was reported to accelerate excisional wound healing in rats. In this study, we investigated the therapeutic effects of ARE on bacterial-infected wounds. Thirty Sprague-Dawley rats were divided into three groups: normal saline (NS), ARE, and biomycin ointment (BO). Full-thickness dorsal excisions in all the rats were infected with 108 colony-forming units of S. aureus; the treatments were applied once daily for 7 days. Results showed that the residual wound area in ARE group was smaller than those in NS and BO groups. TBCs on wound sites gradually decreased in ARE and BO groups. The body temperature and plasma inflammatory cytokines (TNF-α, IL-6) levels increased after bacterial infection at 24 h in all groups. After treatment, BT and inflammatory cytokines levels decreased in ARE group. Histological observations showed ARE group exhibited earlier scab formation, denser dermal granulation tissue, thicker epidermis, and more angiogenesis markers than the other groups. In conclusion, ARE accelerated wound healing in S. aureus-infected wounds. We proposed ARE exhibited potential antimicrobial and anti-inflammatory effects and stimulated angiogenesis, thus improving healing in infected wounds.

Genomic characterization of the intron-containing T7-like phage phiL7 of Xanthomonas campestris.

The lytic phage phiL7, which morphologically belongs to the Siphoviridae family, infects Xanthomonas campestris pv. campestris. Nucleotide sequence analysis has revealed that phiL7 contains a linear double-stranded DNA genome (44,080 bp, 56% G+C) with a 3'-protruding cos site (5'-TTACCGGAC-3') and 59 possible genes. Among the deduced proteins, 32 have homologs with known functions and 18 show no database similarities; moreover, the genes encoding these 18 proteins mostly have varying G+C contents and form clusters dispersed along the genome. Only 39 genes have sequences related (27% to 78%) to those of sequenced genes of X. oryzae pv. oryzae phages, although the genome size and architecture of these Xanthomonas phages are similar. These findings suggest that phiL7 acquired genes by horizontal transfer, followed by evolution via various types of mutations. Major differences were found between phiL7 and the X. oryzae pv. oryzae phages: (i) phiL7 has a group I intron inserted in the DNA polymerase gene, the first such intron observed in Xanthomonas phages; (ii) although infection of phiL7 exerted inhibition to the host RNA polymerase, similar to the situations in X. oryzae pv. oryzae phages Xp10 and Xop411, sequence analysis did not identify a homologue of the Xp10 p7 that controls the shift from host RNA polymerase (RNAP) to viral RNAP during transcription; and (iii) phiL7 lacks the tail fiber protein gene that exhibits domain duplications thought to be important for host range determination in OP1, and sequence analysis suggested that p20 (tail protein III) instead has the potential to play this role.

Effective Treatment of Bovine Mastitis with Intramammary Infusion of Angelica dahurica and Rheum officinale Extracts.

Mastitis in dairy cattle is a highly prevalent infectious disease, causing considerable economic loss worldwide. In this study, we used Angelica dahurica and Rheum officinale extracts (designated as Yi-Xiong-Tang, YXT) for mastitis treatment. California mastitis test (CMT) was performed and 67 mastitis udder quarters were identified among 179 lactating dairy cows. These 67 mastitis udder quarters were subjected to treatments by intramammary infusion of YXT twice a day for three consecutive days. The mastitis indicators including clots, lactate dehydrogenase (LDH), TNF-α, IL-6, IL-8, and total viable count of bacteria (TVC) in milk were examined before and after the YXT treatment to evaluate its effectiveness. Levels of mastitis indicators from mastitis udder quarters were elevated. After YXT treatment, normal levels of these indicators were restored: TVC, 2.10 × 104 - 9.20 × 106 CFU/mL; clots, 6.56 ± 0.43 mg/mL; LDH, 181.0 ± 18.55 U/L; TNF-α, 0.02 ± 0.02 ng/mL; IL-6, 41.4 ± 11.46 pg/mL; and IL-8, 1.85 ± 0.60 pg/mL. Compared with the antibiotic therapy, YXT treatment has a shorter treatment course and might have lower probability for the causative agents to develop drug resistance because YXT is in fact a cocktail containing multiple active ingredients.

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.

Comparison of genomes of three Xanthomonas oryzae bacteriophages.

BACKGROUND: Xp10 and OP1 are phages of Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice plants, which were isolated in 1967 in Taiwan and in 1954 in Japan, respectively. We recently isolated the Xoo phage Xop411. RESULTS: The linear Xop411 genome (44,520 bp, 58 ORFs) sequenced here is 147 bp longer than that of Xp10 (60 ORFs) and 735 bp longer than that of OP1 (59 ORFs). The G+C contents of OP1 (51%) and Xop411 and Xp10 (52% each) are less than that of the host (65%). The 9-bp 3'-overhangs (5'-GGACAGTCT-3') in Xop411 and Xp10 are absent from OP1. More of the deduced Xop411 proteins share higher degrees of identity with Xp10 than with OP1 proteins, while the right end of the genomes of Xp10 and OP1, containing all predicted promoters, share stronger homology. Xop411, Xp10, and OP1 contain 8, 7, and 6 freestanding HNH endonuclease genes, respectively. These genes can be classified into five groups depending on their possession of the HNH domain (HNN or HNH type) and/or AP2 domain in intact or truncated forms. While the HNN-AP2 type endonuclease genes dispersed in the genome, the HNH type endonuclease genes, each with a unique copy, were located within the same genome context. Mass spectrometry and N-terminal sequencing showed nine Xop411 coat proteins, among which three were identified, six were assigned as coat proteins (4) and conserved phage proteins (2) in Xp10. The major coat protein, in which only the N-terminal methionine is removed, appears to exist in oligomeric forms containing 2 to 6 subunits. The three phages exhibit different patterns of domain duplication in the N-terminus of the tail fiber, which are involved in determination of the host range. Many short repeated sequences are present in and around the duplicated domains. CONCLUSION: Geographical separation may have confined lateral gene transfer among the Xoo phages. The HNN-AP2 type endonucleases were more likely to transfer their genes randomly in the genome and may degenerate after successful transmission. Some repeated sequences may be involved in duplication/loss of the domains in the tail fiber genes.

Effects of Angelica dahurica and Rheum officinale Extracts on Excisional Wound Healing in Rats.

The main objective of wound treatments is to restore the functional skin properties and prevent infection. Traditional Chinese medicine provides alternative anti-inflammatory, antimicrobial, and wound healing therapies. Both Angelica dahurica extract (AE) and Rheum officinale extract (RE) possess antimicrobial activity. In this study, AE and RE were applied in wound treatment to investigate their healing effects. Thirty Sprague-Dawley rats with dorsal full-thickness skin excision were divided into normal saline (NS), AE, RE, AE plus RE (ARE), and Biomycin (BM) groups. The treatment and area measurement of wounds were applied daily for 21 days. Wound biopsies and blood samples were obtained for histology examinations and cytokine analysis. Results showed that wound contraction in ARE group was significantly higher than that in NS and BM groups (P < 0.05). Histological analysis showed that more inflammatory cell infiltration, collagen fibers, and myofibroblasts were observed in ARE treated group than those in NS group on days 3-5. In ARE group, plasma IL-6 levels were elevated during days 3-5 (P > 0.05), and plasma TGF-ß1 levels were significantly lower than those in the NS group on days 3-4 (P < 0.05). In conclusion, ARE accelerates wound healing during inflammation and proliferation phases.

Genomic Characterization of the Novel Aeromonas hydrophila Phage Ahp1 Suggests the Derivation of a New Subgroup from phiKMV-Like Family.

Aeromonas hydrophila is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant A. hydrophila isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of A. hydrophila, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous A. hydrophila strains.

Protein-translocating outer membrane porins of Gram-negative bacteria.

Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins.

Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites.

In Xanthomonas campestris, the causative agent of black rot in crucifers, the endoglucanase level is greatly decreased in the mutant deficient in Clp, a homologue of cyclic AMP receptor protein (CRP). It is established that Clp has the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. In this study, the engA transcription initiation site was determined by the 5' RACE method, and two consensus Clp-binding sites, site I and site II centered at -69.5 and -42.5, respectively, were located. Transcriptional fusion assays indicated that Clp greatly activates engA transcription. Site-directed mutagenesis indicated that position 5 of GTG motif in site II is essential for both DNA-protein complex formation in electrophoretic mobility shift assays and engA transcription in vivo. In addition, mutation at position 5 of site I drastically reduces the promoter activity, indicating that binding of Clp to site I exerts a synergistic effect on the transcription activation by site II. engA appears to be the first X. campestris gene known to be activated by Clp via a direct binding to the promoter.

Expression of heat-shock genes groESL in Xanthomonas campestris is upregulated by CLP in an indirect manner.

CLP is a homologue of cyclic AMP-receptor protein in Xanthomonas campestris. In this study, proteomic analysis and Western blotting showed that the clp mutant (TC820) of X. campestris synthesizes less GroESL proteins than the parental P20H. The groESL upstream regions, nt -583 to -32 (552 bp) and nt -178 to -29 (150 bp) relative to the groESL initiation codon, were cloned for transcriptional fusion assays. The 150-bp region, bearing putative sigma24- and sigma32-binding sites and the CIRCE element all known to regulate groESL operon, expressed the same levels of beta-galactosidase (300 U/ml) in both strains, indicating that CLP is not involved in the expression from this region. At early exponential phase, the 552-bp region displayed extremely high levels of promoter activity, 11,000 U/ml in P20H versus 5000 U/ml in TC820. The enzyme levels were about 2000 U/ml at stationary phase in both strains, indicating high levels of expression when cells cease growing. These results suggest that the sequence responding to CLP regulation resides between nt -178 and -583. However, since this region has no CLP-binding site and showed no binding to CLP in gel retardation assay, CLP is likely acting indirectly. This communication appears to be the first description of the positive regulation of a bacterial heat-shock operon by a CRP homologue.

Differential expression of catalases in Vibrio parahaemolyticus under various stress conditions.

Among antioxidant enzymes, catalases protect microorganisms by degrading hydrogen peroxide under oxidative stress. In this study, the activities of at least four Vibrio parahaemolyticus catalases (Kat1 to Kat4) were differentially detected during different growth stages and under various stress conditions using zymographic analysis. Our results showed that only Kat2 is stable at 55 °C. Kat1 and Kat2 respond to hydrogen peroxide during the early stationary and exponential growth phases, respectively and the response decreases upon entering the stationary phase. Kat3 and Kat4 are bifunctional, exhibiting both catalase and peroxidase activities and are only expressed during the stationary phase, under starvation or under stress at pH 5.5. Our study also shows that expression of Kat3 and Kat4 depends on RpoS. We confirm that both monofunctional and bifunctional catalases are expressed and function differentially under various stresses to contribute total catalase activities for the survival of V. parahaemolyticus. A comparative genomic study among Vibrio species revealed that only V. parahaemolyticus contains two copies of genes that encode monofunctional and bifunctional catalases. We propose that both types of catalases, whether evolved or acquired horizontally through long-term evolution, may play crucial protective roles in V. parahaemolyticus in response to environmental fluctuations.

Draft Genome Sequence of Vibrio owensii GRA50-12, Isolated from Green Algae in the Intertidal Zone of Eastern Taiwan.

Vibrio owensii GRA50-12 was isolated from symbiotic green algae of coral. The genome contains genes encoding toxin production, virulence regulation, stress response proteins, types II, IV, and VI secretion systems, and proteins for the metabolism of aromatic compounds, which reflects its pathogenic potential and its ecological roles in the ocean.

Complete Genome Sequence of Xanthomonas campestris pv. campestris Strain 17 from Taiwan.

Xanthomonas campestris pv. campestris 17 is a Gram-negative bacterium that is phytopathogenic to cruciferous plants in Taiwan. The 4,994,426-bp-long genome consists of 24 contigs with 4,050 protein-coding genes, 1 noncoding RNA (ncRNA) gene, 6 rRNA genes, and 55 tRNA genes.

The ubiquitous ThrE family of putative transmembrane amino acid efflux transporters.

We here report sequence analyses of a newly described family of putative amino acid exporters, the ThrE family. Homologues were identified in select bacteria, archaea and eukaryotes, but only in the fungal kingdom of eukaryotes. These proteins can exist either as single polypeptide chains or as pairs of polypeptide chains. Computational evidence suggests that these proteins exhibit 10 transmembrane alpha-helical segments (TMSs), having arisen from a five TMS precursor by an early intragenic duplication event. The phylogenetic tree of the ThrE family reveals that most proteins cluster according to organismal phylogeny with only a few exceptions, suggesting that the former proteins are orthologues. All family members exhibit hydrophilic N-terminal (and occasional C-terminal) extensions that show limited sequence similarity with a domain of unknown function found in many peptidases and proteases. The significance of these observations is discussed.