Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its ...structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2−/− mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.
The structures of two major phosphoglycolipids from the thermophilic bacteria Thermus oshimai NTU-063, Thermus thermophilus NTU-077, Meiothermus ruber NTU-124, and Meiothermus taiwanensis NTU-220 ...were determined using spectroscopic and chemical analyses to be 2′-O-(1,2-diacyl-sn-glycero-3-phospho) -3′-O-(α-N-acetyl-glucosaminyl)-N-glyceroyl alkylamine PGL1 (1) and the novel structure 2′-O-(2-acylalkyldio-1-O-phospho)-3′-O-(α-N-acetylglucosaminyl)-N-glyceroyl alkylamine PGL2 (2). PGL2 (2) is the first phosphoglycolipid identified with a 2-acylalkyldio-1-O-phosphate moiety. The fatty acids of the phosphoglycolipids are mainly iso-C15:0, -C16:0, and -C17:0 and anteiso-C15:0 and -C17:0. The ratios of PGL2 (2) to PGL1 (1) are significantly altered when grown at different temperatures for three strains, T. thermophilus NTU-077, M. ruber NTU-124, and M. taiwanensis NTU-220, but not for T. oshimai NTU-063. Accordingly, the ratios of iso- to anteiso-branched fatty acids increase when grown at the higher temperature.
1 Institute of Biological Chemistry, Academia Sinica, 115 Taipei, Taiwan
2 Institute of Biochemical Sciences, National Taiwan University, 106 Taipei, Taiwan
3 Department of Microbiology, Tzu Chi ...University, 970 Hualien, Taiwan
4 Institute of Plant Biology, National Taiwan University, 106 Taipei, Taiwan
Correspondence San-San Tsay sstsay{at}ntu.edu.tw
Two novel bacteria, with an optimum growth temperature of approximately 60 °C, were isolated from Lu-shan hot springs in the central region of Taiwan. These isolates were aerobic, thermophilic, halotolerant, pink-pigmented, heterotrophic and resistant to gamma-radiation. Both pleomorphic, short, rod-shaped cells and coccoid cells were observed. Strains LS-286 (=ATCC BAA-452=BCRC 17198) and LS-293 T (=ATCC BAA-406 T =BCRC 17173 T ) represented a novel species of the genus Rubrobacter , according to a phylogenetic analysis of the 16S rRNA gene, DNADNA hybridization, biochemical features and fatty acid composition. The name Rubrobacter taiwanensis sp. nov. is proposed for this novel species, with LS-293 T as the type strain.
San-San Tsay and Wen-Chang Chang contributed equally to this work.
Published online ahead of print on 16 April 2004 as DOI 10.1099/ijs.0.63109-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LS-293 T and LS-286 are AF465803 and AF479791 , respectively.
Images produced using scanning electron microscopy and atomic-force microscopy are available, together with pH and salinity data and fatty acid profiles, as supplementary material in IJSEM Online.
The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA‐400 by ethanol extraction and purified by Sephadex LH‐20 and silica gel column chromatography. The ...fatty acid composition of O‐acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C15:0 (34.0%) and C17:0 (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O‐acyl groups under mild basic conditions provided two glycolipids, which differ only in N‐acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C17:0 N‐acyl group and the other hydroxy C17:0 in a ratio of about 1 : 3.5. Furthermore, complete de‐lipidation with strong base followed by selective N‐acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA‐400 was determined with the following structure: α‐Galp(1‐6)‐β‐Galp(1‐6)‐β‐GalNAcyl(1,2)‐α‐Glc(1,1)‐Gro diester, where N‐acyl is C17:0 or hydroxy C17:0 fatty acid and the glycerol esters were mainly iso‐ and anteisobranched C15:0 and C17:0.
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The structure of a major glycolipid isolated from the thermophilic bacteria
Thermus oshimai NTU-063 was elucidated. The sugar and fatty acid compositions were determined by GC–MS and ...HPLC analysis on their methanolysis and methylation derivatives, respectively. After removal of both
O- and
N-acyl groups by alkaline treatment, the glycolipid was converted to a fully acetylated tetraglycosyl glycerol derivative, the structure of which was then determined by NMR spectroscopy (TOCSY, HSQC, HMBC). Thus, the complete structure of the major glycolipid from
T. oshimai NTU-063 was established as β-Glc
p-(1→6)-β-Glc
p-(1→6)-β-Glc
pNAcyl-(1→2)-α-Glc
p-(1→1)-glycerol diester. The
N-acyl groups on the 2-amino-2-deoxy-glucopyranose residue are C
15:0 and C
17:0 fatty acids, whereas the fatty acids of glycerol diester are more heterogeneous including both straight and branched fatty acids from C
15:0 to C
18:0.
The thermophilic bacterium
Thermus aquaticus NTU103 harbors a 1965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames (
...orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from
Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double-stranded origin and single-stranded origin sites, indicating the importance of these
cis elements in pTA103 replication.
d -Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d -ornithine to (2 R ,4 S )-2,4-diaminopentanoic acid. The two genes encoding d -ornithine aminomutase ...have been cloned, sequenced, and expressed in Escherichia coli . The oraS gene, which encodes a protein of 121 amino acid residues with M
r 12,800, is situated upstream of the oraE gene, which encodes a protein of 753 amino acid residues with M
r 82,900. The holoenzyme appears to comprise a α 2 β 2 -heterotetramer. OraS shows no significant homology to other proteins in the Swiss-Prot data base. The deduced amino acid
sequence of OraE includes a conserved base-off/histidine-on cobalamin-binding motif, D X H XX G. OraE was expressed in E. coli as inclusion bodies. Refolding experiments on OraE indicate that the interactions between OraS and OraE and the binding of
either pyridoxal phosphate or adenosylcobalamin play important roles in refolding process. The K m values for d -ornithine, 5â²-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5â²-phosphate (PLP) are 44.5 ± 2.8, 0.43 ± 0.04, and 1.5 ± 0.1
μ m , respectively; the k
cat is 6.3 ± 0.1 s â1 . The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, and d -ornithine being present in the assay; no other cofactors were required. A red-shift in UV-visible absorption spectrum is
observed when free adenosylcobinamide is bound by recombinant d -ornithine aminomutase and no significant change in spectrum when free adenosylcobinamide is bound by mutant OraE-H618G, demonstrating
that the enzyme binds adenosylcobalamin in base-off/histidine-on mode.
A gene encoding thermostable Lon protease from Brevibacillus thermoruber WR‐249 was cloned and characterized. The Br. thermoruber Lon gene (Bt‐lon) encodes an 88 kDa protein characterized by an ...N‐terminal domain, a central ATPase domain which includes an SSD (sensor‐ and substrate‐discrimination) domain, and a C‐terminal protease domain. The Bt‐lon is a heat‐inducible gene and may be controlled under a putative Bacillus subtilisσA‐dependent promoter, but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt‐lon was expressed in Escherichia coli, and its protein product was purified. The native recombinant Br. thermoruber Lon protease (Bt‐Lon) displayed a hexameric structure. The optimal temperature of ATPase activity for Bt‐Lon was 70 °C, and the optimal temperature of peptidase and DNA‐binding activities was 50 °C. This implies that the functions of Lon protease in thermophilic bacteria may be switched, depending on temperature, to regulate their physiological needs. The peptidase activity of Bt‐Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt‐Lon is different from that of E. coli Lon in using fluorogenic peptides as substrates. Notably, the Bt‐Lon protein shows chaperone‐like activity by preventing aggregation of denatured insulin B‐chain in a dose‐dependent and ATP‐independent manner. In thermal denaturation experiments, Bt‐Lon was found to display an indicator of thermostability value, Tm of 71.5 °C. Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt‐Lon relevant to thermostability.
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