Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from Thermotoga maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the E. coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the alpha-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the Thermotoga maritima cofactor-dependent alpha-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.     

Primary structure of the E. coli DNA region preceding the tryptophan operon genes

The nucleotide sequence of 1179 b.p. preceding the trp operon genes has been established. There are no open reading frames large enough to code for proteins containing more than 97 amino acid residues. In all cases the coding sequences do not contain the initiation codons. The determined sequence is concluded to represent an intercistronic region.     

A chromosomal region of Mycoplasma agalactiae containing vsp-related genes undergoes in vivo rearrangement in naturally infected animals

A Mycoplasma agalactiae genomic fragment carrying four vsp-related genes (designated avg: agalactiae variable genes) was cloned, sequenced and compared to the vspA gene of Mycoplasma bovis. The following features were revealed: (i) the presence of a highly conserved vsp 5' upstream region; (ii) a highly homologous vsp N-terminal end encoding a putative lipoprotein signal sequence; (iii) sequence divergence of the rest of the mature proteins. By using avg specific probes in Southern blot analysis of genomic DNAs of M. agalactiae strains as well as of isolates from infected animals, marked DNA polymorphism of avg fragments was demonstrated. In addition, the avg genomic fingerprints were monitored for a period of 7 months, in isolates of M. agalactiae from an individual chronically infected animal. The results provided evidence that a chromosomal region of M. agalactiae, carrying vsp-related genes, undergoes rearrangements in vivo in the natural animal host during the course of infection.     

Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.

Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.     

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.     

The structure and phylogenetic significance of the flagellar transition region in the chlorophyll c-containing algae.

An electron-dense helix is the most conspicuous structure in the flagellar transition region of members of the algal class Chrysophyceae. This “transitional helix” (TH) lies immediately distal to a partition across the flagellar axoneme which occurs exactly at the level at which the flagellum enters the cell body. The helix surrounds the central axonemal pair and lies at a distance of 10 nm from the 9 peripheral doublets. From the new data presented and a survey of published observations on the structure of the transition region of all the chlorophyll c-containing classes of algae, it is shown that a TH characteristic of the Chrysophyceae, Xanthophyceae and Eustigmatophyceae. The number of TH gyres varies from 3 to 6 in the Xanthophyceae and from 1 to 8 in the Chrysophyceae. In any one species, however, the TH is the same size in both the long flagellum which bears tubular mastigonemes and in the short smooth flagellum, though in some chrysophytes where the short flagellum is vestigial the number is fewer than in the normal flagellum. A TH appears to be absent from the Rhaphidophyceae and zoids of the Bacillariophyceae and Phaeophyceae though the structure of the transition region in these groups otherwise resembles that of the Chrysophyceae, Xanthophyceae and Eustigmatophyceae.The value of transition region variation in determining evolutionary relationships among the chlorophyll c-containing algal classes is assessed against a background of current ideas on their taxonomy and phylogeny. The relevant structural and biochemical features are tabled, and a phylogenetic scheme is presented which appears most logically to interpret these data. It is suggested that the line leading to the Eustigmatophyceae probably diverged from that leading to the strictly heterokont classes Xanthophyceae, Chrysophyceae, Phaeophyceae and Bacillariophyceae before evolution of a girdle lamella in the chloroplast and a photoreceptor apparatus involving a swelling at the proximal end of the short flagellum and an intraplastidial eyespot. The possession of a TH by both the Chrysophyceae and Xanthophyceae adds further support to the concept of their close relationship based on a range of other features. The exceptional absence of a TH from the chrysophycean genera Pedinella and Pseudopedinella reinforces the idea that these taxa are remote from the main chrysophycean line. Absence of a TH from the Phaeophyceae and Bacillariophyceae which otherwise share many important features with the Chrysophyceae and Xanthophyceae is probably a result of loss owing to the functional and morphological specialization of the zoids of these two groups. Transition region structure does not clarify the possible relationships of the Rhaphidophyceae, Prymnesiophyceae, Cryptophyceae or Dinophyceae.The proposed phylogeny supports the idea of a mutually related “heterokont” protist assemblage comprising the Chrysophyceae, Xanthophyceae, Phaeophyceae, Bacillariophyceae and possibly Rhaphidophyceae and the Oomycetes (water moulds) though in the latter the TH is replaced by a dense cylinder with a corrugated wall which may or may not be homologous with it. Structures resembling a TH have been described in a wide variety of other flagellated cells including the prasinophyte Pyraminonas orientalis, one species of the colourless flagellate genus Bicosoeca and the proteromonads Karotomorpha and Proteromonas. Only in the latter genera does homology with a TH seem likely on present evidence, suggesting that flagellates of this type may have evolved from chrysomonad-like ancestors.     

Identification of the human chromosomal region containing the iridogoniodysgenesis anomaly locus by genomic-mismatch scanning.

Genome-mismatch scanning (GMS) is a new method of linkage analysis that rapidly isolates regions of identity between two genomes. DNA molecules from regions of identity by descent from two relatives are isolated based on their ability to form extended mismatch-free heteroduplexes. We have applied this rapid technology to identify the chromosomal region shared by two fifth-degree cousins with autosomal dominant iridogoniodysgenesis anomaly (IGDA), a rare ocular neurocristopathy. Markers on the short arm of human chromosome 6p were recovered, consistent with the results of conventional linkage analysis conducted in parallel, indicating linkage of IGDA to 6p25. Control markers tested on a second human chromosome were not recovered. A GMS error rate of approximately 11% was observed, well within an acceptable range for a rapid, first screening approach, especially since GMS results would be confirmed by family analysis with selected markers from the putative region of identity by descent. These results demonstrate not only the value of this technique in the rapid mapping of human genetic traits, but the first application of GMS to a multicellular organism.     

Organization, structure and expression of murine interferon alpha genes.

Using a human interferon-alpha probe we have isolated recombinant phages containing murine interferon-alpha (Mu IFN-alpha) genes from a genomic library. One of these phages contained two complete Mu IFN-alpha genes and part of a third gene. The insert of a second phage held two IFN genes. This indicates that the Mu IFN-alpha genes are clustered in the genome as is the case for the analogous human genes. The nucleotide sequences of these 5 genes were determined. They show that the genes are all different, albeit highly homologous. The deduced amino acid sequences show that four of the five genes contain a putative glycosylation site. Three genes were transiently expressed in COS cells and they gave rise to protein products showing antiviral properties. The expression of the five Mu IFN-alpha genes and the Mu IFN-beta gene was studied in virus-induced mouse L cells. The individual mRNAs were visualized in a nuclease S1 experiment, using a specific probe for each gene. In RNA preparations from induced cells mRNAs for each of the five alpha genes and the beta gene were present. However, substantial differences in the amounts of the individual mRNAs were observed.     

Organization of ribosomal protein genes in Escherichia coli: I. Physical structure of DNA from transducing λ phages carrying genes from the aroE-str region

The physical structures of the genomes of five transducing bacteriophages (λaroE, λtrkA, λspc1, λspc2, and λfus2) carrying various portions of the aroE-trkA-spc-str segment of the Escherichia coli chromosome have been determined. Two methods were used: (a) heteroduplex analysis of DNA molecules from these phages, and (b) analysis of fragments obtained from digestion of the DNA by restriction endonucleases EcoRI and HindIII. In λaroE, λtrkA, λspc1 and λspc2, whose genome lengths vary from about 75% to about 104% of the λpapa genome, the right arm of λ DNA is present, whereas various portions of the left arm have been replaced by E. coli DNA. In λfus2, however, about 93% of the λ DNA molecule is replaced by E. coli DNA, the resultant genome being 103.5 %λ units long (Figs 1 and 2). All five phages contain an identical λ-E. coli junction at 1.9 %λ units from the left λ terminus, and there is complete homology between the common portions of the inserted E. coli DNA. Since these phages were independently isolated, we believe that the genetic organization of the E. coli DNA carried by these phages probably reflects the organization of the relevant segments of the E. coli chromosome. Comparison of the physical and genetic maps of these transducing phages has allowed us to assign a physical position to the ribosomal and neighbouring genes, including those coding for the α subunit of RNA polymerase and the elongation factors G and Tu, on the bacterial DNA.     

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Molecular Structure of the Locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from T. maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the Escherichia coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the -glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the T. maritima cofactor-dependent -glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.