Rituximab therapy for chonic and refractory immune thrombocytopenic purpura : a long-term follow-up analysis The aim of this study was to evaluate the long-term response to rituximab in patients with chronic and refractory immune thrombocytopenic purpura -LRB- ITP -RRB- . Adults with ITP fail to respond to conventional therapies in almost 30 % of cases , developing a refractory disease . Rituximab has been successfully used in these patients . We used rituximab at 375 mg/m2 , IV , weekly for a total of four doses in 18 adult patients . Complete remission -LRB- CR -RRB- was considered if the platelet count was > 100 × 109/l , partial remission -LRB- PR -RRB- if platelets were > 50 × 109/l , minimal response -LRB- MR -RRB- if the platelet count was > 30 × 109/l and < 50 × 109/l , and no response if platelet count remained unchanged . Response was classified as sustained -LRB- SR -RRB- when it was stable for a minimum of 6 months . Median age was 43.5 years -LRB- range , 17 to 70 -RRB- . Median platelet count at baseline was 12.5 × 109/l -LRB- range , 3.0 to 26.3 -RRB- . CR was achieved in five patients -LRB- 28 % -RRB- , PR in five -LRB- 28 % -RRB- , MR in four -LRB- 22 % -RRB- , and two patients were classified as therapeutic failures -LRB- 11 % -RRB- . Two additional patients were lost to follow-up . The median time between rituximab therapy and response was 14 weeks -LRB- range , 4 to 32 -RRB- . SR was achieved in 12 patients -LRB- 67 % -RRB- . There were no severe adverse events during rituximab therapy . During follow-up -LRB- median , 26 months ; range , 12 to 59 -RRB- , no other immunosuppressive drugs were used . In conclusion , rituximab therapy is effective and safe in adult patients with chronic and refractory ITP . Overall response rate achieved is high , long term , and with no risk of adverse events . Introduction In immune thrombocytopenic purpura -LRB- ITP -RRB- , platelets are coated with an IgG autoantibody that prompts its premature destruction and , as a result , different grades of peripheral thrombocytopenia and clinical bleeding become evident -LSB- 5 , 23 -RSB- . Steroids are the first-line treatment for acute ITP . In case of failure to steroids , splenectomy induces a 70 to 80 % response rate -LSB- 10 -RSB- . However , almost 30 % of adults with ITP fail to respond to conventional therapies -LSB- steroids , IV immunoglobulin -LRB- IVIg -RRB- , splenectomy , or immunosuppressive drugs -RSB- and , eventually , they develop a chronic refractory disease -LSB- 5 , 12 , 23 -RSB- . Refractory patients are defined as those who failed standard-dose steroids and splenectomy , requiring further treatment due to unsafe platelet counts -LRB- < 30 × 109/l -RRB- or clinical bleeding . These patients are unlikely to be cured , although spontaneous remissions sometimes occur -LSB- 17 -RSB- . If a patient becomes refractory , some alternatives are available such as danazol , dapsone , azathioprine , high-dose steroids , or chemotherapy combinations like CHOP regimen . However , these options are not quite useful because if response is achieved it is but for a brief period and sustained responses are scarce -LSB- 23 -RSB- . Hematopoietic stem cell transplantation has been occasionally used in some cases , but this option represents a high-risk procedure -LSB- 14 -RSB- . At the end , these patients may respond poorly to subsequent therapy , may have significant morbidity from the disease and its treatment -LRB- bleeding and infections secondary to immunosuppressive therapy -RRB- , and the mortality rate at 10 years may rise up to 20 % -LSB- 5 , 11 -RSB- . On the other hand , these patients have higher hemorrhagic risk imposed by trauma or surgery . Therefore , new treatment strategies for these patients are needed . Rituximab is an anti-CD20 chimeric murine-human monoclonal antibody . CD20 is a B-lymphocyte membrane protein absent on other normal cells . This monoclonal IgG-kappa antibody is used in non-Hodgkin lymphomas because it induces apoptosis or direct lysis of B cells -LSB- 19 -RSB- . In ITP , B cells are responsible of autoantibody production and the subsequent platelet opsonization that allows its destruction . Therefore , if B cell clones are eliminated , thrombocytopenia could be reverted . There are some reports informing that rituximab is useful in refractory ITP showing a high overall response rate , with complete remission -LRB- CR -RRB- and partial remission -LRB- PR -RRB- rates ranging from 48 to 54 % -LSB- 4 , 8 , 9 , 18 , 20 -- 22 -RSB- . We report herein our long-term experience with rituximab therapy in chronic and refractory ITP . Materials and methods In a 5-year period , we prospectively administered rituximab to all patients with chronic and refractory ITP who were unresponsive to prednisone , azathioprine , vincristine , danazol , and high-dose dexametasone , among other therapies , including those unsuccessfully splenectomized . Patients must have a platelet count of < 30 × 109/l to be considered for this therapy . Rituximab was given in doses of 375 mg/m2 , diluted in 500 ml 5 % dextrose -- water solution in a 4-h continuous IV infusion . This regimen was indicated weekly for four doses after prophylactic application of antipyretic and antihistamine premedication administered 30 min before rituximab infusion -LRB- paracetamol , 500 mg , PO ; diphenhydramine , 30 mg , PO ; and hydrocortisone , 100 mg , IV -RRB- . Outpatient follow-up was scheduled monthly for 6 months . Long-term follow-up was performed every 2 months . Response was classified as complete -LRB- CR -RRB- if the platelet count was > 100 × 109/l , partial -LRB- PR -RRB- if platelet count > 50 × 109/l , minimal response -LRB- MR -RRB- if platelet count was < 50 × 109/l and > 30 × 109/l , and no response -LRB- NR -RRB- if there was no change from the baseline platelet count . Response was sustained -LRB- SR -RRB- when it was maintained for a minimum of 6 months . Numerical variables were expressed using the median and range number . The probability to achieve more than 50 × 109/l and 100 × 109/l platelets and the median time from response to end of follow-up was calculated using the Kaplan and Meier method . Before starting treatment , all patients signed an informed consent authorization . The procedures done in this protocol were in accordance with the Helsinki Declaration of 1975 . The protocol was approved by the Ethics Committee of the participating hospitals . Results Eighteen patients were candidates for this therapy , and all of them were suitable for evaluation because they received the planned four doses of rituximab . Patient characteristics and response to rituximab are shown in Table 1 . Median age for the whole group was 43.5 years -LRB- range , 17 to 70 -RRB- . Median platelet count at baseline was 12.5 × 109/l -LRB- range , 3.0 to 26.3 -RRB- . Median follow-up was 26 months -LRB- range , 12 to 59 -RRB- . Mean number of treatment regimens received before rituximab was 5.5 -LRB- range , 3 to 8 -RRB- . Fifteen patients -LRB- 83 % -RRB- had failed to splenectomy . Table 1Patient characteristics and outcomesPatient/age/genderEvolutiona -LRB- months -RRB- Previous treatmentsPC at baseline -LRB- × 109/l -RRB- PC at 12 months -LRB- × 109/l -RRB- Time to response -LRB- weeks -RRB- Response typeSR1/42/F37P , S , Az , D , IFN3 .014624 CRYes2/43/F84P , S , Az , D , IFN , V5 .012324 CRYes3/30/F48P , S , Az , D , IFN , V12 .534 . 516MRYes4b/27/F38P , S , Az , D , IFN24 .47816 PRbYesb5/70/M60P , Az , D , V7 .8 NRNRNRNo6/43/F96P , S , Az , D , IFN , V10 .137.78 MRYes7/53/F96P , S , Az , D , IFN , V12 .55412 PRYes8/36/F264P , S , Az , D , IFN , V , OE , IVIg5 .030.64 MRYes9/17/F38P , S , Az , D , IFN , V , OE6 .51294 CRYes10/57/F26P , Az , D8 .3648 PRYes11/36/F25P , S , Az , D , IFN , V7 .04920 MRNo12/63/F120P , Az , D , V26 .3 NRNRNRNo13/53/F348P , De , D , S , Az22 .01128 CRYes14/22/F266P , S , D , IFN , IVIg12 .66712 PRNo15/56/F137P , D , S , Az18 .42234 CRYes16/35/F38P , D , Az , S23 .211.40 NRNo17/52/F41P , D , S16 .551.94 PRYes18/24/F60O , D , S24 .0 NRNRNRNoPC Platelet count , P prednisone , S splenectomy , Az azathioprine , D danazol , IFN interferon alpha 2b , V vincristine , De dexamethasone , OE opsonized erythrocytesaTime from ITP diagnosis to rituximab therapybA second course of rituximab was given . CR was achieved in five patients -LRB- 28 % -RRB- , PR was obtained in another five -LRB- 28 % -RRB- , MR was seen in four -LRB- 22 % -RRB- patients , and two patients were considered treatment failures -LRB- 11 % -RRB- . Two patients were lost to follow-up -LRB- 11 % -RRB- . Ten patients -LRB- 55 % -RRB- achieved > 50 × 109/l platelets . Median time from the first rituximab dose to achievement of any response was 14 weeks -LRB- range , 4 to 32 -RRB- . Twelve patients -LRB- 67 % -RRB- showed SR beyond the sixth month of follow-up . As expected from the results depicted in Table 1 , we were unable to find any correlation between the ITP duration and the type of response to rituximab . Figure 1 shows the median platelet counts during follow-up in three different groups of patients classified according to their pattern of response -LRB- CR , PR , and MR -RRB- . The median time to achieve > 50 × 109/l platelets was 5 months -LRB- 95 % CI = 0.5 to 11.6 months -RRB- , and the median time to achieve > 100 × 109/l platelets was not reached -LRB- Fig. 2 -RRB- . One patient relapsed 22 months after the first course of rituximab . She received steroids for another 14 months ; she did not obtain response , and a second course of rituximab was given using the regimen described previously . She achieved a new PR 6 months after stopping therapy . After 22 months of follow-up , PR still remains in this patient . Fig. 1Platelet count achieved after first dose of rituximab -LRB- time 0 first dose of rituximab -RRB- . Diamonds , CR ; ovals , PR ; triangles , MRFig . 2Time required to obtain platelet counts > 50 × 109/l -LRB- a -RRB- or > 100 × 109/l -LRB- b -RRB- after first dose of rituximab in adult patients with chronic and refractory ITP Median time of response duration was 54 months -LRB- 95 % CI = 15 -- 93 months -RRB- for patients with CR , 18 months -LRB- 95 % CI = 8 -- 28 months -RRB- for patients with partial response , and 12 months -LRB- 95 % CI = 7 -- 17 months -RRB- for those individuals with minimal response . Difference was statistically significant between patients in CR vs those achieving PR or MR -LRB- p < 0.05 -RRB- . No difference was observed between PR and MR -LRB- Fig. 3 -RRB- . Fig. 3Median duration of response for patients who achieved complete remission -LRB- CR ; 54 months , 95 % CI = 15 -- 93 months -RRB- , partial remission -LRB- PR ; 18 months , 95 % CI = 8 -- 28 months -RRB- , or minor response -LRB- MR ; 12 months , 95 % CI = 7 -- 17 months -RRB- Side effects related to the first dose of rituximab such as fever , chills , and respiratory symptoms were common -LRB- 8 out of 18 patients , 43 % -RRB- . There were no severe adverse events during drug administration . Neither hemorrhagic events nor infections were recorded . Neutropenia , an occasional long-term side effect of rituximab therapy , was never recorded in our group of patients -LRB- Fig. 4 -RRB- . In fact , the lower neutrophil count recorded during the study was 1.3 × 109/l in patient 7 after 6 weeks of treatment with rituximab . No patient needed additional immunosuppressive therapy other than rituximab . During the whole follow-up period , no other illnesses have been recorded in this group of patients . Fig. 4Mean -LRB- squares -RRB- , maximum -LRB- diamonds -RRB- , and minimum -LRB- triangles -RRB- neutrophil counts through the follow-up of patients receiving rituximab for chronic and refractory ITP Discussion Because almost 30 % of individuals with ITP do not respond to the first - and second-line therapies , they live with low platelet counts , a situation that carries a high risk of hemorrhage and , eventually , a short life expectancy . It has been largely demonstrated that ITP with persistent low platelet counts carries a grave prognosis -LSB- 6 , 13 -RSB- . Efforts to increase the platelet count to at least 30 × 109/l include IVIg , anti-D immunoglobulin , cyclophosphamide , polychemotherapy such as the CHOP regimen , thrombopoietin , interleukin-11 , dapsone , alpha interferon , plasma exchange , and bone marrow transplantation -LSB- 23 -RSB- . Using these therapies , the response rate is low and the patient is exposed to unnecessary risks -LSB- 10 -RSB- . When the first ITP patient successfully treated with rituximab was informed , it drew the attention of physicians because this regimen offered high response rate and low toxicity . In subsequent reports , researchers have found similar results . The aim of this study was to evaluate the response rate achieved in 18 patients with chronic and refractory ITP after rituximab treatment as well as the evolution of these patients to learn about the possible long-term side effects associated with the use of this drug , information that is almost inexistent in the literature . We observed 12 favorable responses -LRB- 67 % -RRB- and at least a stable clinical evolution in two additional patients -LRB- 11 % -RRB- , for a global response rate of 78 % . Based on the characteristics of the evolution of our group of patients , we may emphasize some points of interest : -LRB- 1 -RRB- Response rate obtained was quite satisfactory considering the history of chronicity and refractoriness of the patients ; -LRB- 2 -RRB- the monoclonal antibody was well tolerated and caused only mild allergic reactions that could be easily managed with antihistaminics and paracetamol ; -LRB- 3 -RRB- most of the patients achieve SR ; and -LRB- 4 -RRB- increase of platelet count was not always immediate -LRB- median time to response = 14 weeks -RRB- , a fact that slightly disagrees with previous reports that found shorter time to response -LRB- 3 to 8 weeks ; Table 2 -RRB- -LSB- 4 , 8 , 20 -RSB- . In fact , in a recent systematic review about the efficacy of rituximab in adult patients with ITP , it was found that median time to response was 5.5 weeks -LRB- ranges 2 to 18 weeks -RRB- -LSB- 2 -RSB- . We do not have an explanation for this discrepancy . After a careful analysis , we did not find significant differences between the results obtained in the systematic review and our results in terms of the variables that may affect the time to response to rituximab , namely , age , sex , previous use of corticosteroids , number of treatments before rituximab , rituximab dose and schedule , period between diagnosis and rituximab administration , duration of ITP before rituximab , and pretreatment platelet counts . Although splenectomy may not be a significant predictor of response to rituximab as previously suggested -LSB- 2 , 4 , 8 -RSB- , it should be noted that 83 % of the patients included in our report had had splenectomy as compared with 50.5 % of the patients in the systematic review . Therefore , history of splenectomy seems to be the only factor related to a different time to response to rituximab in our series . Table 2Patterns of response in patients with chronic and refractory ITP -LRB- from -LSB- 1 , 6 , 18 -RSB- -RRB- ReferenceNumber of patientsOR -LRB- % -RRB- CR -LRB- % -RRB- PR -LRB- % -RRB- Median time to response -LRB- weeks -RRB- Mayo clinic1250428?USA -- Italy575331228Denmark353318152 -- 8Peru22684027 ? Mexico1856282814OR Overall response , CR complete response , PR partial response Of course , we have no data about all possible variables influencing the pattern of response to rituximab . For example , we do not show information about B-cell counts and platelet autoantibodies before and after rituximab therapy , two variables that may influence the response as observed in other trials . The expected therapeutic effect of rituximab is a reduction in specific platelet-associated autoantibodies and the consequent increase in platelet counts . Although rituximab has not been always associated with a reduced load of platelet autoantibodies and most of the publications reporting this effect are small series of cases -LSB- 1 -RSB- , the high overall response rate obtained in our study allows us to believe that rituximab effectively decreased the B-cell counts as well as the levels of platelet autoantibodies . Three patterns of response to rituximab have been proposed : early -LRB- before the fourth dose of rituximab -RRB- , intermediate -LRB- 7 to 11 weeks after rituximab -RRB- , and delayed -LRB- > 13 weeks after rituximab -RRB- -LSB- 8 -RSB- . In our study , the probability to achieve a platelet count > 50 × 109/l occurred at a median of 5 months , so we speculate that the complete inhibition of antibody formation and restoration of platelet counts with rituximab may occur after at least 5 months -LRB- 95 % CI = 0.5 to 11.6 months -RRB- from the first dose of the antibody -LRB- Fig. 2 -RRB- . However , some patients may achieve a quite delayed response -LRB- as long as 1 year after therapy -RRB- , a situation in which it is important to wait a reasonable time period before another treatment is planned . A previous report informed the results obtained in patients treated with the same regimen as we used -LSB- 4 -RSB- . After a median follow-up of 47 weeks , the authors observed a lower overall response rate of 44 % -LRB- CR = 18 % , PR = 15 % , MR = 10 % -RRB- ; most of the responses were sustained . They observed two response patterns : -LRB- 1 -RRB- an early-response group in which responses appeared within the first 2 weeks after the first dose of rituximab ; -LRB- 2 -RRB- a late-response group characterized by an increase in platelets several weeks after rituximab . Finally , after a median follow-up of 72.5 weeks , the response rate was 54 % with a majority of SR -LSB- 8 -RSB- . In a prospective trial performed in pediatric patients with chronic ITP , treatment with rituximab produced an increase of > 50 × 109/l platelets in 11 of 36 children -LRB- 31 % -RRB- -LSB- 3 -RSB- . Median time to response was 1 week -LRB- range , 1 to 7 weeks -RRB- ; however , a 6 % incidence of serum sickness was observed . More recently , one of the largest experiences in ITP patients treated with rituximab was published -LSB- 15 -RSB- . An increase of > 50 × 109/l platelets was observed in 55 % of the patients -LRB- CR = 46 % , SR = 35 % -RRB- . The only predictive factor for SR was to achieve CR . Patients who were treated more intensively -LRB- more than three different treatments -RRB- and those with a longer ITP duration -LRB- > 10 years from diagnosis -RRB- had the worse response . In this study , non-splenectomized patients had a higher early response rate than those splenectomized . Although some of these results are similar to those reported in our study , we believe that contrasting results seen in our study are partially explained by differences in the inclusion and response criteria used , in the evolution time of the disease , and in the number and type of treatments given before rituximab . As early relapses may be seen in ITP patients treated with rituximab and because re-treatment with this monoclonal antibody offers good results -LSB- 16 -RSB- , this drug has been used as a long-term maintenance treatment . Based on long-lasting remissions achieved using one or two doses of rituximab -LSB- 7 -RSB- and the results of late responders , we feel that a single dose of 375 mg/m2 every 6 months could be an alternative for this purpose . On the other hand , because of the mechanism of action of rituximab and its interference with the immune system , we searched for possible long-term complications associated with its use , namely , neoplasias , chronic infections , or autoimmune diseases . Immune status is a major concern in patients treated with rituximab being the induction of low CD20 + counts and hypogammaglobulinemia -LRB- specifically low levels of immunoglobulin M -RRB- , two previously reported secondary effects . Although we do not have data about these two immunological variables , the lack of severe infectious diseases during the long-term follow-up of our patients allows us to speculate that no severe immune abnormalities were developed in our series . As we previously stated , during the follow-up period , no associated illnesses or pathological phenomena were observed . To our knowledge , this seems to be the first report about the lack of long-term complications associated with rituximab . In conclusion , rituximab is a long-term , safe , and effective alternative treatment of chronic and refractory ITP patients . Prospective randomized clinical trials are needed to elucidate the efficacy of the drug in comparison to splenectomy in early stages of the disease

B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O


Structural

characterization

of

diabolic

acid-based

tetraester

tetraether

and

mixed

ether/ester

membrane-spanning

lipids

of

bacteria

from

the

order

Thermotogales

The

distribution

of

core

lipids

in

the

membranes

of

nine

different

species

of

the

order

Thermotogales

one

of

the

early

and

deep

branching

lineages

in

the

Bacteria

were

examined

by

HPLC/MS

and

demonstrated

to

consist

of

membrane-spanning

diglycerol

lipids

comprised

of

diabolic

acid-derived

alkyl

moieties

In

the

Thermotoga

species

the

core

membrane

lipids

are

characterized

by

the

presence

of

both

ester

and

ether

bonds

whereas

in

the

phylogenetically

more

distinct

Thermosipho

and

Fervidobacterium

spp.

only

ester

bonds

occur

A

tentative

biosynthetic

route

for

the

biosynthesis

of

these

membrane-spanning

lipids

is

proposed

Since

species

of

the

order

Thermotogales

are

assumed

to

have

occurred

early

during

the

evolution

of

life

on

Earth

as

suggested

by

its

position

in

the

phylogenetic

tree

of

life

these

data

suggest

that

the

ability

to

produce

both

ether

and

ester

glycerol

membrane

lipids

developed

relatively

early

during

microbial

evolution

Introduction

The

group

of

anaerobic

bacteria

falling

in

the

order

Thermotogales

represents

a

very

deep

phylogenetic

branch

within

the

phylogenetic

tree

of

life

based

upon

the

ribosomal

RNA

gene

-LRB-

Woese

-RRB-

Within

this

order

members

of

the

genus

Thermotoga

have

an

upper

growth

limit

of

°C

and

represent

together

with

species

in

the

order

Aquificales

cultivated

bacteria

with

the

highest

known

growth

temperatures

Bacteria

belonging

to

the

Thermotogales

occur

widespread

in

the

environment

they

thrive

within

continental

solfatara

springs

of

low

salinity

shallow

and

deep-sea

marine

hydrothermal

systems

and

high-temperature

marine

and

continental

oil

fields

-LRB-

see

Huber

and

Hannig

for

a

recent

review

-RRB-

Their

strict

organotrophic

way

of

life

makes

them

consumers

of

microbial

organic

matter

within

high

temperature

ecosystems

Within

the

order

Thermotogales

the

genera

Thermotoga

Thermosipho

Fervidobacterium

Geotoga

Petrotoga

and

Marinitoga

have

been

described

all

belonging

to

the

single

family

Thermotogaceae

-LRB-

Huber

and

Hannig

-RRB-

The

type

species

T.

maritima

for

the

genus

Thermotoga

also

the

first

identified

member

of

the

Thermotogales

was

isolated

from

a

geothermally

heated

shallow

marine

sediment

in

Italy

-LRB-

Huber

et

al.

-RRB-

Subsequently

quite

a

number

of

phylogenetically

closely

related

-LRB-

T.

neapolitana

T.

petrophila

T.

naphtophila

-RRB-

and

slightly

more

distant

-LRB-

T.

thermarum

T.

elfii

T.

subterranea

T.

hypogea

T.

lettingae

-RRB-

species

were

isolated

mostly

from

marine

settings

Thermotoga-related

organisms

have

been

detected

in

oil

reservoirs

-LRB-

Huber

and

Hannig

-RRB-

whilst

members

of

the

genus

Fervidobacterium

seem

to

be

restricted

to

hydrothermal

settings

with

low

salinity

such

as

hot

springs

For

example

Fervidobacterium

pennivorans

was

firstly

isolated

from

a

hot

spring

on

San

Miguel

-LRB-

the

Azores

-RRB-

-LRB-

Friedrich

and

Antranikian

-RRB-

Members

of

the

genus

Thermosipho

have

been

isolated

from

a

wide

variety

of

high

temperature

environments

such

as

marine

hydrothermal

springs

-LRB-

e.g.

T.

africanus

-RRB-

and

deep

sea

hydrothermal

vents

-LRB-

e.g.

T.

melanesiensis

-RRB-

-LRB-

Antoine

et

al.

Huber

et

al.

-RRB-

To

be

able

to

grow

at

high

temperatures

the

members

of

the

Thermotogales

possess

a

variety

of

thermostable

enzymes

and

proteins

which

are

also

of

biotechnological

interest

In

addition

the

cell

membranes

of

these

organisms

must

be

able

to

cope

with

high

temperatures

Indeed

a

variety

of

unusual

membrane

lipids

has

been

reported

for

members

of

the

Thermotogales

Unusual

long-chain

dicarboxylic

-LRB-

diabolic

-RRB-

acids

are

present

in

the

core

lipids

of

the

genus

Thermotoga

-LRB-

Jeanthon

et

al.

Manca

et

al.

Windberger

et

al.

Carballeira

et

al.

DeRosa

et

al.

Huber

et

al.

-RRB-

About

of

the

total

polar

lipids

of

T.

maritima

are

two

amphipathic

monopolar

glycolipids

with

a

very

rare

α

-LRB-

-RRB-

diglucosyl

structure

-LRB-

Manca

et

al.

-RRB-

In

the

present

paper

we

describe

in

detail

the

structures

of

these

and

other

lipids

in

a

suite

of

bacteria

belonging

to

the

Thermotogales

and

discuss

their

distributions

We

applied

an

HPLC/MS

method

which

for

the

first

time

enabled

to

characterize

the

full

membrane-spanning

core

lipids

Materials

and

methods

Cultures

Cells

were

grown

in

batch

cultures

in

at

temperatures

of

°C

and

with

either

medium

A

or

B

-LRB-

see

Table

-RRB-

Medium

A

was

composed

-LRB-

per

liter

demineralized

water

-RRB-

of

KCl

g

g

g

g

g

NaCl

g

glucose

g

yeast

extract

g

peptone

g

g

g

trace

element

solution

ml

and

vitamin

solution

ml

which

were

based

on

medium

of

DSM

-LRB-

-RRB-

g

resazurin

mg

The

medium

was

anaerobically

dispensed

into

serum

bottles

and

a

gas

phase

of

kPa

-LRB-

v/v

-RRB-

was

applied

glucose

and

vitamin

solution

were

added

after

sterilization

The

pH

was

Medium

B

is

a

modified

DSM

medium

and

composed

-LRB-

per

liter

demineralized

water

-RRB-

of

g

NaCl

g

g

g

g

peptone

g

yeast

extract

g

trace

element

solution

-LRB-

see

DSM

medium

-RRB-

ml

mg

glucose

g

cysteine

HCl

g

resazurin

mg

The

medium

was

anaerobically

dispensed

into

serum

bottles

and

a

gas

phase

of

kPa

-LRB-

-RRB-

was

applied

Glucose

and

cysteine

HCl

were

added

after

sterilization

The

pH

was

adjusted

to

pH

The

cultures

were

inoculated

with

cells

from

the

DSM

collection

of

microorganisms

-LRB-

see

Table

for

strain

number

-RRB-

and

were

harvested

in

their

exponential

growth

phase

Two

different

batches

of

T.

lettingae

were

grown

and

analysed

separately

Table

from

the

order

Thermotogales

with

some

background

information

and

the

culture

conditions

used

in

this

studyBacterial

speciesSource

for

the

isolateOptimal

growth

temperature

-LRB-

°C

-RRB-

ReferenceDSM

strain

numberCulture

conditionsMediumaT

-LRB-

°C

-RRB-

Thermotoga

maritimaAnaerobic

marine

sediment

Italy

et

al.

-LRB-

-RRB-

neapolitanaSubmarine

hot

spring

Italy

et

al.

-LRB-

-RRB-

elfiiOil-producing

well

Africa

et

al.

-LRB-

-RRB-

lettingaeSulfate-reducing

bioreactor

The

Netherlands

et

al.

-LRB-

-RRB-

hypogeaOil-producing

well

Africa

et

al.

-LRB-

-RRB-

sp.Spring

New

Zealand

africanusMarine

hydrothermal

spring

Obock

et

al.

-LRB-

-RRB-

melanesiensisDeep

sea

hydrothermal

vent

Pacific

Ocean

et

al.

-LRB-

-RRB-

pennivoransHot

mud

of

spa

Italy

et

al.

-LRB-

-RRB-

See

Materials

and

method

sections

for

a

description

of

the

media

used

Lipid

analysis

Lyophilized

cells

were

repeatedly

-LRB-

-RRB-

ultrasonically

extracted

with

dichloromethane

-LRB-

DCM

-RRB-

methanol

-LRB-

MeOH

-RRB-

-LRB-

v/v

-RRB-

The

solvent

of

the

combined

extracts

was

removed

by

rotary

evaporation

at

reduced

pressure

The

extract

was

taken

up

in

DCM

and

weighed

in

a

small

vial

by

removing

the

solvent

under

a

gentle

stream

of

nitrogen

An

aliquot

-LRB-

ca.

mg

-RRB-

of

the

extract

to

which

a

known

amount

of

the

internal

standard

was

added

was

methylated

with

filtered

over

a

small

pipette

filled

with

silica

using

ethyl

acetate

as

the

eluent

and

subsequently

silylated

with

N,O-bis

-LRB-

trimethylsilyl

-RRB-

trifluoroacetamide

-LRB-

BSTFA

-RRB-

in

pyridine

at

°C

for

min

These

so-called

total

lipid

fractions

-LRB-

-RRB-

were

analysed

with

gas

chromatography

-LRB-

GC

-RRB-

and

gas

chromatography

mass

spectrometry

-LRB-

GC

MS

-RRB-

In

selected

cases

acetylation

instead

of

silylation

of

the

eluate

obtained

after

chromatography

over

silica

was

performed

following

a

procedure

described

elsewhere

-LRB-

Cox

et

al.

-RRB-

The

cell

residue

after

solvent

extraction

was

after

addition

of

a

known

amount

of

the

internal

standard

hydrolysed

with

N

KOH/MeOH

-LRB-

-RRB-

by

refluxing

for

h

The

hydrolysate

was

neutralized

with

N

HCl/MeOH

and

after

addition

of

water

extracted

with

DCM

This

fraction

was

methylated

chromatographed

over

silica

and

silylated

as

described

above

and

the

resulting

fraction

was

analyzed

by

GC

and

GC

MS.

In

specific

cases

alkaline

hydrolysis

was

also

performed

with

an

aliquot

of

the

solvent

extract

following

the

same

procedure

as

described

above

This

resulted

in

a

fraction

named

which

was

also

analyzed

by

GC

and

GC

MS.

For

analysis

of

core

lipids

by

high

performance

liquid

chromatography/atmospheric

pressure

chemical

ionization

mass

spectrometry

-LRB-

HPLC/APCI

MS

-RRB-

aliquots

of

the

solvent

extracts

-LRB-

ca.

mg

-RRB-

were

separated

over

a

small

column

using

hexane/DCM

-LRB-

v/v

-RRB-

and

DCM/MeOH

as

eluent

to

obtain

apolar

and

polar

fractions

respectively

The

polar

fraction

was

dissolved

in

hexane/propanol

-LRB-

v/v

-RRB-

filtered

over

an

μm

PTFE

filter

and

analysed

by

HPLC/APCI

MS.

GC

and

GC

MS

GC

was

performed

using

a

Fisons

instrument

equipped

with

an

on-column

injector

and

a

flame

ionization

detector

A

fused

silica

capillary

column

-LRB-

m

mm

-RRB-

coated

with

CP

-LRB-

df

um

-RRB-

was

used

with

helium

as

carrier

gas

The

samples

were

injected

at

°C

and

the

oven

temperature

was

programmed

to

°C

at

°C

min

and

then

at

°C

min

to

°C

at

which

it

was

held

for

min

GC

MS

was

performed

on

a

HP

gas

chromatograph

interfaced

with

a

VG

Autospec

Ultima

mass

spectrometer

operated

at

eV

with

a

mass

range

of

m/z

and

a

cycle

time

of

s

-LRB-

resolution

-RRB-

The

gas

chromatograph

was

equipped

with

a

fused

silica

capillary

column

as

described

for

GC

The

carrier

gas

was

helium

The

same

temperature

program

as

for

GC

was

used

HPLC/APCI

MS

Membrane-spanning

core

lipids

were

analysed

by

HPLC/APCI

MS

according

to

Hopmans

et

al.

-LRB-

-RRB-

with

minor

modifications

Analyses

were

performed

on

an

Agilent

series

HPLC/MS

instrument

equipped

with

an

auto-injector

and

HP

Chemstation

software

Separation

was

achieved

on

an

Alltech

Prevail

Cyano

column

-LRB-

mm

mm

μm

-RRB-

Flow

rate

of

the

hexane

propanol

-LRB-

v/v

-RRB-

eluent

was

ml

min

isocratically

for

the

first

min

thereafter

with

a

linear

gradient

to

propanol

in

min

subsequently

to

propanol

in

min

and

subsequently

isocratically

for

the

last

min

Injection

volume

of

the

samples

was

usually

μl

Results

Nine

species

of

bacteria

belonging

to

the

order

Thermotogales

were

analysed

for

their

lipid

composition

five

of

them

are

Thermotoga

species

-LRB-

T.

maritima

T.

lettingae

T.

elfii

T.

neapolitana

T.

hypogea

-RRB-

three

are

Thermosipho

species

-LRB-

T.

africanus

T.

melanesiensis

T.

sp.

-RRB-

and

one

is

a

Fervidobacterium

species

-LRB-

F.

pennivorans

-RRB-

-LRB-

Table

-RRB-

Composition

of

total

lipid

fractions

Figure

shows

a

typical

gas

chromatogram

of

a

total

lipid

fraction

-LRB-

-RRB-

for

T.

maritima

In

addition

to

straight-chain

fatty

acids

-LRB-

predominantly

and

-RRB-

a

number

of

more

unusual

lipids

were

detected

The

first

group

comprises

long-chain

dicarboxylic

acids

acid

-LRB-

or

diabolic

acid

see

Fig.

for

structures

-RRB-

and

acid

-LRB-

-RRB-

identified

on

basis

of

mass

spectral

data

reported

previously

-LRB-

Carballeira

et

al.

Klein

et

al.

-RRB-

The

mass

spectra

of

and

as

methyl

ester

derivatives

are

shown

in

Fig.

The

relative

abundance

of

the

and

diabolic

acids

varied

substantially

between

different

species

-LRB-

Table

-RRB-

F.

pennivorans

also

contained

small

amounts

of

a

tentatively

identified

diabolic

acid

In

some

species

isomers

of

these

diabolic

acids

with

virtually

an

identical

mass

spectrum

but

with

a

different

retention

time

were

present

in

varying

quantities

These

probably

represent

stereoisomers

possibly

related

to

the

presence

of

two

chiral

centres

in

the

middle

of

the

carbon

chain

of

the

diabolic

acids

The

second

group

of

unusual

components

comprises

glycerol

monoethers

glycerol

-LRB-

-RRB-

and

glycerol

-LRB-

-RRB-

were

identified

by

mass

spectral

data

-LRB-

m/z

base

peak

and

appropriate

molecular

ion

-RRB-

and

a

co-elution

experiment

with

an

authentic

standard

The

higher

molecular

weight

monoethers

are

15,16-dimethyl-30-glyceryloxytriacontanoic

acid

-LRB-

-RRB-

and

13,14-dimethyl-28-glyceryloxyoctadecanoic

acid

-LRB-

-RRB-

Glycerol

monoether

has

been

identified

previously

and

the

mass

spectrum

of

as

acetyl

derivative

was

reported

-LRB-

DeRosa

et

al.

-RRB-

For

identification

purposes

we

used

acetylation

in

addition

to

silylation

to

be

able

to

compare

the

mass

spectrum

of

the

derivative

of

with

that

reported

in

the

literature

-LRB-

DeRosa

et

al.

-RRB-

The

mass

spectrum

of

as

TMS

derivative

is

shown

in

Fig.

Component

was

tentatively

identified

by

interpretation

of

its

mass

spectrum

in

comparison

with

that

of

-LRB-

Fig.

-RRB-

The

relative

amounts

of

monoethers

and

in

the

vary

widely

between

the

examined

species

of

the

Thermotogales

-LRB-

Table

-RRB-

As

observed

for

the

diabolic

acids

and

two

additional

minor

isomers

with

similar

mass

spectrometric

characteristics

but

different

retention

times

are

observed

The

third

group

of

lipids

are

glycerol

dialkyl

diesters

and

glycerol

mixed

ester/ethers

They

were

predominantly

found

in

the

of

T.

lettingae

-LRB-

Fig.

-RRB-

and

are

composed

of

and

glycerols

-LRB-

e.g.

and

-RRB-

and

-LRB-

e.g.

-RRB-

and

-LRB-

e.g.

-RRB-

glycerols

They

were

identified

by

comparison

with

mass

spectra

reported

in

the

literature

-LRB-

Wood

et

al.

-RRB-

Some

Thermotoga

species

contained

small

amounts

of

glycerols

Fig.

chromatograms

of

the

total

lipid

fractions

-LRB-

-RRB-

-LRB-

a

b

-RRB-

and

these

fractions

after

alkaline

hydrolysis

of

the

residue

after

extraction

-LRB-

-RRB-

-LRB-

c

d

-RRB-

of

Thermotoga

maritima

-LRB-

grown

at

°C

-RRB-

and

F.

pennivorans

pennivorans

respectively

Fatty

acids

and

alcohols

were

derivatized

to

the

corresponding

methyl

esters

and

trimethylsilyl

ethers

prior

to

gas

chromatographic

analysis

Numbers

refer

to

structures

indicated

in

Fig.

FA

Fatty

acid

std

internal

standardFig

of

lipids

encountered

in

species

from

the

order

ThermotogalesFig

spectra

-LRB-

subtracted

for

background

-RRB-

of

derivatised

diabolic

acids

and

diabolic

acid

glycerol

ethersTable

distribution

-LRB-

in

of

total

quantified

-RRB-

of

lipids

in

different

species

of

the

order

ThermotogalesBacterial

maritima

-LRB-

°C

-RRB-

T.

maritima

-LRB-

°C

-RRB-

T.

neapolitanaTLF-14.661.32.817.31.33.70.77.11.2

T.

T.

lettingae

-LRB-

batch

-RRB-

T.

lettingae

-LRB-

batch

-RRB-

T.

hypogeaTLF-14.854.52.314.12.37.43.58.13.0

Thermosipho

T.

T.

Fervidobacterium

a

Total

lipid

fraction

total

lipid

fraction

obtained

after

alkaline

hydrolysis

of

the

residue

after

extraction

TLF-OH

Total

lipid

fraction

after

alkaline

hydrolysis

of

the

extractb

Numbers

refer

to

Fig.

Blank

entries

indicate

that

the

lipid

was

not

detectedFig

chromatograms

of

the

total

lipid

fraction

-LRB-

-RRB-

-LRB-

a

-RRB-

this

fraction

after

alkaline

hydrolysis

-LRB-

-RRB-

-LRB-

b

-RRB-

and

this

fraction

obtained

after

alkaline

hydrolysis

of

the

residue

after

extraction

-LRB-

-RRB-

-LRB-

c

-RRB-

of

Thermotoga

lettingae

-LRB-

batch

-RRB-

Fatty

acids

and

alcohols

were

derivatized

to

the

corresponding

methyl

esters

and

trimethylsilyl

ethers

prior

to

gas

chromatographic

analysis

Numbers

refer

to

structures

indicated

in

Fig.

FA

Fatty

acid

std

internal

standard

The

TLF

after

alkaline

hydrolysis

of

the

cell

residue

after

extraction

-LRB-

-RRB-

contained

a

similar

suite

of

lipids

as

the

-LRB-

Table

-RRB-

albeit

that

some

other

components

-LRB-

e.g.

unsaturated

and

fatty

acids

-RRB-

were

also

present

but

only

in

the

examined

Thermosipho

species

Most

striking

was

that

the

characteristic

long-chain

diacids

and

and

the

complex

monoethers

and

were

relatively

much

more

abundant

in

-LRB-

Figs.

d

and

Table

-RRB-

suggesting

that

they

are

predominantly

formed

by

alkaline

hydrolysis

from

larger

lipid

structures

A

similar

distribution

was

observed

when

the

extract

of

T.

lettingae

-LRB-

batch

-RRB-

was

subjected

to

alkaline

hydrolysis

-LRB-

-RRB-

the

long-chain

diacids

and

and

the

monoethers

and

which

were

not

observed

in

the

-LRB-

Fig.

-RRB-

are

one

of

the

most

abundant

lipids

in

the

fraction

-LRB-

Fig.

-RRB-

This

indicates

that

these

lipids

are

building

blocks

of

larger

more

complex

lipids

not

amenable

to

GC

The

absolute

amount

of

the

most

abundant

lipids

-LRB-

in

mg

g

dry

weight

-RRB-

was

for

a

number

of

species

measured

in

both

the

and

-LRB-

Table

-RRB-

This

showed

that

the

amounts

released

by

alkaline

hydrolysis

from

the

residue

after

extraction

are

still

substantial

It

also

showed

that

the

highest

concentrations

occur

in

the

Thermotoga

species

and

in

F.

pennivorans

while

concentrations

in

the

Thermosipho

species

are

substantially

lower

Quantification

of

the

lipids

in

the

fraction

of

T.

lettingae

-LRB-

batch

-RRB-

showed

that

the

concentration

increases

substantially

after

alkaline

hydrolysis

of

the

extract

Table

concentrations

-LRB-

in

mg

g

dry

weight

-RRB-

of

diabolic

acids

and

glycerol

monoether

acids

in

different

species

of

the

order

ThermotogalesBacterial

maritima

-LRB-

°C

-RRB-

T.

lettingae

-LRB-

batch

-RRB-

T.

Thermosipho

T.

T.

a

Total

lipid

fraction

total

lipid

fraction

obtained

after

alkaline

hydrolysis

of

the

residue

after

extraction

total

lipid

fraction

after

base

hydrolysis

of

the

extractb

Numbers

refer

to

Fig.

Blank

entries

indicate

that

the

lipid

was

not

detected

Distribution

of

membrane-spanning

core

lipids

in

T.

maritima

To

characterize

the

membrane-spanning

core

lipids

of

the

species

belonging

to

the

Thermotogales

the

polar

fraction

of

the

solvent

extract

was

analysed

by

an

HPLC/APCI

MS

method

-LRB-

Hopmans

et

al.

-RRB-

slightly

adjusted

to

enable

the

elution

of

more

polar

components

than

glycerol

dialkyl

glycerol

tetraethers

-LRB-

GDGTs

-RRB-

from

the

LC

column

-LRB-

see

Materials

and

methods

for

details

-RRB-

Analysis

of

the

polar

lipids

of

Thermotogamaritima

revealed

a

complex

composition

exemplified

by

the

base

peak

chromatogram

-LRB-

Fig.

-RRB-

The

first

eluting

small

peak

with

a

retention

time

of

min

-LRB-

labelled

in

Fig.

-RRB-

showed

an

APCI

mass

spectrum

-LRB-

Fig.

-RRB-

characterised

by

peaks

at

-LSB-

M

H

-RSB-

-LSB-

M

H

-RSB-

-LRB-

loss

of

water

-RRB-

and

-LSB-

M

H

-RSB-

-LRB-

loss

of

glycerol

-RRB-

This

characteristic

pattern

is

well

known

from

archaeal

GDGTs

-LRB-

Hopmans

et

al.

-RRB-

and

non-isoprenoidal

GDGTs

-LRB-

Sinninghe

Damsté

et

al.

-RRB-

suggesting

a

tetraether

structure

Indeed

upon

alkaline

hydrolysis

of

this

fraction

and

re-analysis

by

HPLC/APCI

MS

this

minor

peak

was

now

the

most

abundant

peak

in

the

base

peak

chromatogram

These

data

indicate

a

GDGT

structure

for

the

major

peak

comprised

of

two

glycerol

units

connected

by

two

alkyl

chains

resulting

in

a

molecular

mass

of

-LRB-

-RRB-

Since

acid

-LRB-

-RRB-

and

acid

-LRB-

-RRB-

are

abundant

lipids

in

the

of

T.

maritima

it

seems

likely

that

the

alkyl

chains

in

this

GDGT

are

the

same

resulting

in

structure

At

the

flank

of

peak

at

slightly

higher

retention

time

a

similar

mass

spectrum

was

observed

with

all

ions

shifted

by

Da

to

lower

m/z

values

This

compound

is

likely

a

GDGT

with

a

and

dimethyl

alkyl

chains

-LRB-

-RRB-

consistent

with

the

presence

of

small

amounts

of

acid

-LRB-

-RRB-

and

acid

-LRB-

-RRB-

in

the

-LRB-

Table

-RRB-

Fig.

MS

base

peak

chromatogram

-LRB-

a

-RRB-

and

specific

mass

chromatograms

-LRB-

b

i

-RRB-

of

polar

lipids

of

Thermotoga

maritima

grown

at

°C

The

mass

chromatograms

indicative

for

specific

components

are

m/z

-LRB-

b

-LSB-

M

H

-RSB-

tetraether

-RRB-

m/z

-LRB-

c

-LSB-

M

H

-RSB-

triether/monoester

-RRB-

m/z

-LRB-

d

-LSB-

M

H

-RSB-

triether/monoester

-RRB-

m/z

-LRB-

e

-LSB-

M

H

-RSB-

diether/diester

-RRB-

m/z

-LRB-

f

-LSB-

M

H

-RSB-

diether/diester

-RRB-

m/z

-LRB-

g

-LSB-

M

H

-RSB-

monoether/triester

-RRB-

m/z

-LRB-

h

-LSB-

M

H

-RSB-

monoether/triester

-RRB-

m/z

-LRB-

i

-LSB-

M

H

-RSB-

tetraester

-RRB-

m/z

-LRB-

j

-LSB-

M

H

-RSB-

tetraester

-RRB-

Filled

and

blocked

peaks

indicate

membrane-spanning

lipids

based

on

and

on

branched

alkyl

chains

respectively

Note

that

some

mass

chromatograms

are

shown

twice

as

they

reveal

the

presence

of

different

componentsFig

APCI

mass

spectra

-LRB-

corrected

for

background

-RRB-

of

-LRB-

a

-RRB-

the

tetraether

-LRB-

b

-RRB-

the

triether/monoester

or

-LRB-

c

-RRB-

the

diether/diester

-LRB-

d

-RRB-

the

ether/triester

-LRB-

e

-RRB-

the

diester

-LRB-

f

-RRB-

the

diether/diester

The

first

larger

peak

in

the

base

peak

chromatogram

at

a

retention

time

of

min

-LRB-

labelled

or

in

Fig.

-RRB-

shows

an

APCI

mass

spectrum

-LRB-

Fig.

-RRB-

in

which

all

ions

are

shifted

by

Da

to

higher

m/z

values

relative

to

those

in

the

mass

spectrum

of

GDGT

-LRB-

Fig.

-RRB-

In

addition

the

-LSB-

M

H

-RSB-

peak

is

substantially

increased

relative

to

the

-LSB-

M

H

-RSB-

peak

and

an

-LSB-

M

H

-RSB-

peak

is

also

observed

Since

this

peak

disappears

when

the

fraction

is

subjected

to

alkaline

hydrolysis

this

component

is

likely

to

contain

an

ester

moiety

This

information

together

with

its

molecular

mass

indicates

that

this

component

is

structurally

related

to

GDGT

but

with

one

of

the

ether

bonds

replaced

by

an

ester

bond

a

glycerol

dialkyl

glycerol

triether/monoester

with

two

dimethyl

alkyl

chains

-LRB-

-RRB-

resulting

in

a

molecular

mass

of

-LRB-

-RRB-

The

presence

of

the

ester

moiety

likely

results

in

the

enhanced

-LSB-

M

H

-RSB-

peak

in

its

APCI

mass

spectrum

Mass

chromatography

of

m/z

-LRB-

Fig.

-RRB-

the

abundant

-LSB-

M

H

-RSB-

peak

reveals

that

there

is

an

additional

much

later

eluting

isomer

with

an

identical

mass

spectrum

This

could

relate

to

the

position

of

the

ester

moiety

in

this

type

of

components

with

the

parallel

configuration

of

the

glycerol

groups

as

indicated

in

there

are

two

possible

isomers

-LRB-

and

-RRB-

However

it

is

at

present

unclear

why

these

positional

isomers

would

differ

to

a

large

degree

in

polarity

-LRB-

i.e.

the

second

isomer

elutes

much

later

and

is

thus

much

more

polar

than

the

first

one

-RRB-

At

the

flank

of

the

peak

of

the

triether/monoester

a

component

elutes

with

a

mass

spectrum

similar

to

that

of

but

with

all

the

major

ions

shifted

by

Da

to

lower

m/z

values

This

component

is

the

triether/monoester

with

and

dimethyl

alkyl

chains

-LRB-

-RRB-

Mass

chromatography

of

m/z

-LRB-

Fig.

-RRB-

the

abundant

-LSB-

M

H

-RSB-

peak

of

this

component

reveals

a

second

much

later

eluting

isomer

for

this

component

as

well

The

most

abundant

peak

in

the

base

peak

ion

chromatogram

at

a

retention

time

of

min

-LRB-

Fig.

-RRB-

shows

a

mass

spectrum

similar

to

that

of

the

triether/monoester

but

in

which

again

all

major

ions

are

shifted

by

Da

to

higher

m/z

values

-LRB-

cf.

Fig.

and

c

-RRB-

This

component

likely

represents

a

glycerol

dialkyl

glycerol

diether/diester

with

two

dimethyl

alkyl

chains

-LRB-

-RRB-

resulting

in

a

molecular

mass

of

m/z

-LRB-

-RRB-

and

an

-LSB-

M

H

-RSB-

of

m/z

This

component

is

composed

of

two

esterified

acid

-LRB-

-RRB-

units

Moreover

mass

chromatography

of

m/z

-LRB-

Fig.

-RRB-

reveals

the

presence

of

a

diester/diether

with

and

building

blocks

-LRB-

-RRB-

Both

the

mass

chromatograms

of

m/z

-LRB-

Fig.

-RRB-

and

m/z

-LRB-

Fig.

-RRB-

reveal

the

presence

of

a

much

later

eluting

isomer

with

similar

mass

spectra

This

latter

cluster

of

peaks

contains

co-eluting

components

with

a

mass

spectrum

with

peaks

at

m/z

and

presumably

the

corresponding

glycerol

dialkyl

glycerol

monoether/triester

with

two

dimethyl

alkyl

chains

-LRB-

-RRB-

Mass

chromatograms

of

m/z

-LRB-

Fig.

-RRB-

and

m/z

-LRB-

Fig.

-RRB-

show

the

presence

of

a

monoether/triester

with

two

dimethyl

alkyl

moieties

-LRB-

-RRB-

and

and

building

blocks

-LRB-

-RRB-

and

much

later

eluting

isomers

for

both

components

These

isomers

possess

identical

mass

spectra

-LRB-

e.g.

Fig.

-RRB-

Finally

small

amounts

of

tetraesters

with

two

dimethyl

alkyl

moieties

-LRB-

-RRB-

and

a

and

a

dimethyl

alkyl

moiety

were

detected

by

mass

chromatography

of

m/z

-LRB-

Fig.

-RRB-

and

m/z

-LRB-

Fig.

-RRB-

respectively

A

typical

mass

spectrum

is

shown

in

Fig.

In

these

traces

three

distinct

isomers

were

observed

The

distribution

of

membrane-spanning

lipids

in

T.

maritima

is

not

depending

on

the

growth

temperature

cells

grown

at

and

°C

showed

almost

the

same

distribution

Distribution

of

membrane-spanning

core

lipids

in

T.

lettingae

The

distribution

of

the

membrane-spanning

lipids

in

T.

lettingae

was

quite

different

-LRB-

Fig.

-RRB-

from

that

observed

in

T.

maritima

it

was

dominated

by

the

diether/diesters

with

the

components

with

two

building

blocks

-LRB-

m/z

Fig.

-RRB-

and

those

with

a

and

a

dimethyl

alkyl

chain

-LRB-

m/z

Fig.

-RRB-

in

approximately

equal

abundance

Diether/diesters

with

two

building

blocks

are

present

in

relatively

small

amounts

as

revealed

by

the

m/z

mass

chromatogram

-LRB-

Fig.

-RRB-

This

is

consistent

with

the

substantially

higher

abundance

of

the

diabolic

acid

-LRB-

-RRB-

relative

to

the

diabolic

acid

-LRB-

-RRB-

and

the

glycerol

monoether

relative

to

the

glycerol

monoether

in

the

fraction

of

T.

lettingae

when

compared

to

T.

maritima

-LRB-

Table

-RRB-

The

relatively

high

abundance

of

lipids

with

a

and

a

dimethyl

and

to

a

lesser

extent

with

two

dimethyl

alkyl

chains

is

also

observed

for

the

monoether/triesters

-LRB-

Fig.

g

-RRB-

and

tetraesters

-LRB-

Figs.

j

-RRB-

Tetraethers

-LRB-

e.g.

-RRB-

which

were

present

in

small

quantities

in

T.

maritima

were

not

encountered

in

T.

lettingae

Fig.

MS

base

peak

chromatogram

-LRB-

a

-RRB-

and

specific

mass

chromatograms

-LRB-

b

j

-RRB-

of

polar

lipids

of

Thermotoga

lettingae

The

mass

chromatograms

indicative

for

specific

components

are

m/z

-LRB-

b

-LSB-

M

H

-RSB-

diether/diester

-RRB-

m/z

-LRB-

c

-LSB-

M

H

-RSB-

diether/diester

-RRB-

m/z

-LRB-

d

-LSB-

M

H

-RSB-

diether/diester

-RRB-

m/z

-LRB-

e

-LSB-

M

H

-RSB-

monoether/triester

-RRB-

m/z

-LRB-

f

-LSB-

M

H

-RSB-

monoether/triester

-RRB-

m/z

-LRB-

g

-LSB-

M

H

-RSB-

monoether/triester

-RRB-

m/z

-LRB-

h

-LSB-

M

H

-RSB-

tetraester

-RRB-

m/z

-LRB-

i

-LSB-

M

H

-RSB-

tetraester

-RRB-

and

m/z

-LRB-

j

-LSB-

M

H

-RSB-

tetraester

-RRB-

Filled

blocked

and

grey

peaks

indicate

membrane-spanning

lipids

based

on

and

branched

alkyl

chains

respectively

Note

that

some

mass

chromatograms

are

shown

twice

as

they

reveal

the

presence

of

different

components

An

additional

cluster

of

early

eluting

peaks

not

observed

in

the

HPLC/MS

analysis

of

the

extract

of

T.

maritima

is

present

in

the

base

peak

chromatogram

of

the

polar

fraction

of

T.

lettingae

-LRB-

Fig.

-RRB-

The

mass

spectra

of

the

peaks

comprising

this

cluster

are

characterized

by

one

dominant

fragment

ion

which

is

Da

higher

than

that

of

the

-LSB-

M

H

-RSB-

peak

of

the

diether/diesters

-LRB-

cf.

Fig.

c

-RRB-

Mass

chromatography

of

m/z

and

-LRB-

Fig.

d

-RRB-

reveals

that

this

cluster

of

peaks

is

build

up

in

a

similar

way

as

the

clusters

reflecting

the

diether/diesters

ether/triesters

and

tetraesters

-LRB-

cf.

Fig.

-RRB-

These

mass

chromatograms

also

reveal

that

these

compounds

are

accompanied

by

a

later

eluting

isomer

which

possess

similar

mass

spectra

These

components

likely

represent

glycerol

trialkyl

glycerol

diether/diesters

-LRB-

-RRB-

comprising

one

long-chain

or

a

dimethyl

alkyl

chain

and

two

shorter

linear

-LRB-

predominantly

-RRB-

alkyl

chains

It

is

well

known

from

archaeal

GDGT

analyses

that

so-called

open

or

acyclic

-LRB-

in

comparison

to

the

macrocyclic

components

described

so

far

-RRB-

GDGTs

elute

substantially

earlier

under

these

LC

conditions

-LRB-

and

are

thus

less

polar

-RRB-

and

possess

APCI

mass

spectra

that

do

not

show

an

-LSB-

M

H

-RSB-

ion

resulting

from

the

loss

of

a

glycerol

moiety

-LRB-

Hopmans

et

al.

-RRB-

Instead

they

easily

loose

one

of

the

shorter

alkyl

chains

upon

APCI

Indeed

when

the

polar

fraction

of

T.

lettingae

was

analysed

using

an

extended

mass

range

-LRB-

m/z

-RRB-

an

abundant

ion

at

m/z

was

observed

which

is

likely

formed

by

loss

of

a

moiety

Mass

chromatography

of

ions

Da

higher

than

used

to

detect

the

monoether/triesters

and

tetraesters

reveals

the

presence

of

acyclic

monoether/triesters

-LRB-

Fig.

g

-RRB-

and

tetraesters

-LRB-

Fig.

j

-RRB-

Fig.

MS

base

peak

chromatogram

-LRB-

a

-RRB-

and

specific

mass

chromatograms

-LRB-

b

j

-RRB-

of

polar

lipids

of

Thermotoga

lettingae

The

mass

chromatograms

indicative

for

specific

components

are

m/z

-LRB-

b

-LSB-

M

H

-RSB-

open

diether/diester

-RRB-

m/z

-LRB-

c

-LSB-

M

H

-RSB-

open

diether/diester

-RRB-

m/z

-LRB-

d

-LSB-

M

H

-RSB-

open

diether/diester

-RRB-

m/z

-LRB-

e

-LSB-

M

H

-RSB-

open

monoether/triester

-RRB-

m/z

-LRB-

f

-LSB-

M

H

-RSB-

open

monoether/triester

-RRB-

m/z

-LRB-

g

-LSB-

M

H

-RSB-

monoether/triester

-RRB-

m/z

-LRB-

h

-LSB-

M

H

-RSB-

open

tetraester

-RRB-

m/z

-LRB-

i

-LSB-

M

H

-RSB-

open

tetraester

-RRB-

and

m/z

-LRB-

j

-LSB-

M

H

-RSB-

open

tetraester

-RRB-

for

the

polar

fraction

of

Thermotoga

lettingae

Black

blocked

and

grey

peaks

indicate

membrane-spanning

lipids

based

on

and

branched

alkyl

chains

respectively

Note

that

some

mass

chromatograms

are

shown

twice

as

they

reveal

the

presence

of

different

components

Distribution

of

membrane-spanning

core

lipids

in

F.

pennivorans

Membrane-spanning

lipids

were

also

detected

in

F.

pennivorans

-LRB-

data

not

shown

-RRB-

In

this

case

the

distribution

was

relatively

simple

only

tetraesters

with

predominantly

two

dimethyl

alkyl

chains

-LRB-

-RRB-

were

detected

in

a

distribution

similar

to

those

observed

in

T.

maritima

-LRB-

Fig.

-RRB-

and

T.

lettingae

-LRB-

Fig.

-RRB-

i.e.

dominated

by

three

isomers

Smaller

amounts

of

tetraesters

with

a

and

a

dimethyl

alkyl

chain

were

also

detected

In

addition

the

also

contained

a

glycerol

esterified

with

two

acid

moieties

or

acid

and

acid

Discussion

Our

results

indicate

that

the

diabolic

acids

and

their

ether

derivatives

previously

identified

in

members

of

the

Thermotogales

-LRB-

DeRosa

et

al.

Windberger

et

al.

Jeanthon

et

al.

Huber

et

al.

-RRB-

are

synthesized

to

produce

membrane-spanning

lipids

and

probably

represent

biosynthetic

intermediates

The

relative

concentration

of

the

diabolic

acids

and

their

ether

derivatives

is

substantially

higher

in

the

extract

obtained

after

alkaline

hydrolysis

of

the

extracted

cell

residue

-LRB-

-RRB-

-LRB-

Table

-RRB-

The

HPLC/APCI

MS

results

clearly

revealed

the

presence

and

in

some

cases

the

structural

complexity

of

the

membrane-spanning

core

lipids

in

species

of

the

order

Thermotogales

for

the

first

time

Previous

investigators

-LRB-

Clarke

et

al.

-RRB-

have

suggested

that

diabolic

acids

may

act

as

linkers

between

two

glycerol

moieties

in

polar

membrane

lipids

of

Butyrivibrio

spp.

It

is

generally

thought

that

the

production

of

membrane-spanning

lipids

in

prokaryotes

is

an

adaptation

to

high

growth

temperature

although

Butyrivibrio

is

a

mesophile

The

ubiquitous

presence

of

diabolic

acid-based

membrane-spanning

lipids

in

members

of

the

Thermotogales

which

are

all

-LRB-

hyper

-RRB-

thermophiles

is

in

line

with

this

general

idea

-LRB-

Table

-RRB-

However

the

distribution

of

membrane-spanning

lipids

in

T.

maritima

is

not

directly

affected

by

the

temperature

of

growth

at

both

and

°C

diabolic

acid-based

membrane-spanning

lipids

were

produced

Despite

the

overall

similarity

in

the

lipid

composition

there

is

a

clear

dichotomy

all

members

of

the

genus

Thermotoga

produce

ether

lipids

-LRB-

i.e.

-RRB-

in

addition

to

ester

lipids

whereas

ether

lipids

are

absent

in

the

genera

Thermosipho

and

Fervidobacterium

-LRB-

Table

-RRB-

For

example

the

HPLC/APCI

MS

results

showed

only

the

presence

of

tetraesters

in

F.

pennivorans

whereas

the

Thermotoga

species

investigated

showed

a

wide

variety

of

mixed

ester/ether

membrane

spanning

core

lipids

-LRB-

Figs.

-RRB-

This

suggests

that

only

Thermotoga

species

possess

enzymes

to

produce

ether

linkages

This

classification

based

on

lipid

biochemistry

is

in

good

agreement

with

the

phylogenetic

classification

based

on

the

sequence

of

the

rRNA

gene

-LRB-

Fig.

-RRB-

which

reveals

that

the

genera

Thermosipho

and

Fervidobacterium

are

quite

distinct

from

the

genus

Thermotoga

although

the

phylogenetic

diversity

within

the

Thermotoga

cluster

is

also

substantial

Fig.

tree

based

on

the

rRNA

gene

outlining

the

relationship

between

molecular

phylogeny

and

lipid

composition

for

the

studied

species

of

the

Thermotogales

The

neighbor-joining

tree

was

reconstructed

from

distance

matrices

and

Escherichia

coli

served

as

outgroup

The

bar

indicates

an

evolutionary

distance

of

Since

no

rRNA

gene

sequence

is

available

for

F.

penniforans

two

other

Fervidobacterium

species

were

used

in

the

construction

of

the

tree

Gene

Bank

accession

numbers

are

given

in

parentheses

The

composition

of

the

low-molecular-weight

lipids

-LRB-

i.e.

fatty

acids

glycerol

monoethers

and

-RRB-

higher

molecular-weight

lipids

-LRB-

-RRB-

and

the

membrane-spanning

lipids

suggest

a

biosynthetic

relationship

between

these

different

lipid

classes

although

the

relatively

high

concentrations

of

lipids

also

suggest

a

direct

functional

role

It

has

been

demonstrated

that

diabolic

acids

are

produced

from

condensation

reactions

of

fatty

acids

at

the

positions

-LRB-

Fitz

and

Arigoni

-RRB-

These

authors

showed

by

the

use

of

-LSB-

-RSB-

palmitic

acid

and

-LSB-

-RSB-

palmitic

acid

that

unsaturated

fatty

acids

are

involved

in

this

condensation

reaction

and

that

it

may

be

mediated

by

a

free

radical

reaction

In

good

agreement

with

the

proposed

biosynthetic

pathway

for

diabolic

acids

is

that

in

all

species

investigated

in

our

study

the

fatty

acid

is

the

major

fatty

acid

and

acid

-LRB-

-RRB-

composed

of

two

condensed

fatty

acid

moieties

is

the

major

diabolic

acid

-LRB-

Table

-RRB-

In

cases

where

the

and

-LRB-

-RRB-

diabolic

acids

are

higher

in

concentration

-LRB-

e.g.

F.

pennivorans

and

T.

lettingae

and

Thermosipho

sp.

respectively

-RRB-

also

a

concomitant

increase

of

the

required

monomer

i.e.

the

and

fatty

acids

respectively

is

observed

-LRB-

Table

-RRB-

This

is

also

seen

in

the

presumed

biosynthetic

end-products

i.e.

the

membrane-spanning

core

lipids

For

example

T.

lettingae

contains

relatively

more

of

these

lipids

based

on

-LRB-

-RRB-

diabolic

acids

whereas

in

F.

pennivorans

the

diabolic

acid

is

a

more

important

building

block

This

is

consistent

with

the

published

observation

that

during

growth

of

T.

maritima

in

batch

culture

the

concentration

of

the

fatty

acid

decreased

with

a

concomitant

increase

of

tentatively

identified

diabolic

acid

-LRB-

Zhang

et

al.

-RRB-

This

was

interpreted

to

reflect

the

result

of

synthesis

of

diabolic

acid

using

the

fatty

acid

The

same

observations

can

be

made

for

the

ether

lipids

only

encountered

in

the

Thermotoga

species

The

presence

of

mono

alkyl

glycerol

ethers

in

all

Thermotoga

species

-LRB-

Table

-RRB-

and

mixed

glycerol

ether/esters

in

T.

lettingae

-LRB-

batch

-RRB-

-LRB-

Fig.

-RRB-

suggests

that

formation

of

the

ether

bond

may

also

occur

before

the

condensation

reaction

resulting

in

the

synthesis

of

membrane-spanning

core

lipids

takes

place

In

archaea

GDGTs

are

also

thought

to

be

produced

from

-LRB-

partial

-RRB-

condensation

of

two

glycerol

diethers

-LRB-

Nishihara

et

al.

Kon

et

al.

Koga

et

al.

-RRB-

Generally

the

distribution

of

the

membrane-spanning

lipids

is

quite

complex

in

the

species

from

the

Thermotogales

It

is

noteworthy

that

several

isomers

with

a

quite

different

polarity

were

detected

in

the

HPLC

traces

The

structural

composition

of

the

diacyl

glycerols

and

the

mixed

glycerol

ester/ethers

in

T.

lettingae

perhaps

provides

a

clue

to

the

observed

complex

composition

of

the

membrane-spanning

core

lipids

Our

analyses

revealed

that

the

ester

moiety

in

these

lipids

can

be

at

both

at

position

and

of

the

glycerol

unit

when

the

ether

or

ester

moiety

is

at

position

-LRB-

Fig.

-RRB-

It

is

quite

likely

-LRB-

although

this

remains

to

be

proven

-RRB-

that

this

also

holds

for

membrane-spanning

core

lipids

This

introduces

a

wide

variety

of

structural

isomers

which

may

explain

why

more

than

one

peak

was

detected

in

the

various

HPLC

mass

chromatograms

-LRB-

Figs.

and

-RRB-

Our

data

show

that

some

members

of

the

Thermotogales

-LRB-

i.e.

the

Thermotoga

genus

-RRB-

produce

membrane-spanning

lipids

which

are

comprised

of

mixed

glycerol

ether/ester

derivatives

whereas

all

examined

species

produce

membrane-spanning

diabolic

acids

Mixed

glycerol

ether/ester

lipids

have

been

increasingly

recognized

in

the

Bacterial

Kingdom

e.g.

in

Planctomycetes

capable

of

anaerobic

ammonium

oxidation

-LRB-

Sinninghe

Damsté

et

al.

Sinninghe

Damsté

et

al.

-RRB-

in

deep-branching

thermophilic

bacteria

such

as

Aquifex

-LRB-

Huber

et

al.

-RRB-

and

in

two

mesophilic

sulfate-reducing

bacteria

-LRB-

Rütters

et

al.

-RRB-

The

tetraethers

and

which

occur

in

low

amounts

in

T.

maritima

are

to

the

best

of

our

knowledge

the

first

example

of

this

kind

of

lipid

in

bacteria

Non-isoprenoidal

GDGTs

have

been

detected

before

in

peats

-LRB-

Sinninghe

Damsté

et

al.

-RRB-

but

their

biological

origin

is

as

yet

unknown

Membrane

spanning

lipids

are

quite

common

in

the

Kingdom

Archaea

-LRB-

Koga

et

al.

-RRB-

but

have

also

been

identified

in

a

very

limited

number

of

bacteria

e.g.

in

Butyrivibrio

spp.

-LRB-

Clarke

et

al.

-RRB-

and

in

Thermoanaerobacter

species

-LRB-

Jung

et

al.

-RRB-

It

is

tempting

to

associate

the

peculiar

membrane

biochemistry

of

the

order

Thermotogales

to

its

position

in

the

phylogenetic

tree

of

life

i.e.

close

to

the

root

of

the

tree

-LRB-

Woese

et

al.

-RRB-

The

unusually

numerous

archaeal-like

genes

-LRB-

almost

-RRB-

found

in

the

Thermotoga

maritima

genome

-LRB-

Nelson

et

al.

-RRB-

reflect

that

these

are

deep-branching

bacteria

If

species

from

the

order

Thermotogales

occurred

early

during

the

evolution

of

life

on

Earth

as

suggested

by

its

position

in

the

phylogenetic

tree

of

life

our

results

indicate

that

the

ability

to

produce

both

ether

and

ester

glycerol

membrane

lipids

developed

relatively

early

during

evolution

All

Archaea

produce

ether

membrane

lipids

whereas

Bacteria

and

Eukaryotes

typically

produce

glycerol

esters

Some

members

of

the

Thermotogales

thus

represent

a

hybrid

in

this

way

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

diabolic acid
ether lipids
ester lipids
thermotoga
thermosipho
fervidobacterium


SSRscanner : a program for reporting distribution and exact location of simple sequence repeats Simple sequence repeats -LRB- SSRs -RRB- have become important molecular markers for a broad range of applications , such as genome mapping and characterization , phenotype mapping , marker assisted selection of crop plants and a range of molecular ecology and diversity studies . These repeated DNA sequences are found in both prokaryotes and eukaryotes . They are distributed almost at random throughout the genome , ranging from mononucleotide to trinucleotide repeats . They are also found at longer lengths -LRB- > 6 repeating units -RRB- of tracts . Most of the computer programs that find SSRs do not report its exact position . A computer program SSRscanner was written to find out distribution , frequency and exact location of each SSR in the genome . SSRscanner is user friendly . It can search repeats of any length and produce outputs with their exact position on chromosome and their frequency of occurrence in the sequence . Background SSRs -LRB- simple sequence repeats -RRB- or microsatellites are the genetic loci where one or few bases are tandemly repeated for varying numbers of times . Such repetitions occur primarily due to slipped-strand mis-pairing and subsequent error -LRB- s -RRB- during DNA replication , repair , or recombination . -LSB- 1 -RSB- SSRs comprising 16 bp long , occur frequently and are ubiquitously interspersed in many genomes . -LSB- 2 -- 3 -RSB- The biological importance of SSR tracts has been clearly delineated . Microsatellite loci show extensive length polymorphism , and hence they are widely used in DNA fingerprinting and diversity studies . They are also considered as ideal genetic markers for the construction of high-density linkage maps . -LSB- 4 -- 5 -RSB- In spite of its high significance , a bioinformatics tool for the analysis of these regions is not available . Available algorithms directly or indirectly detect tandem repeats . However , there are many limitations with these algorithms . The drawbacks are high computational time required by the algorithm and their inability to predict the positions of SSRs in the genome . The program Tandem Repeats Finder -LSB- 6 -RSB- locates repeats with motifs of any size and type , including repeats with insertions and deletions . The program Sputnik -LSB- 7 -RSB- -LRB- unpublished -RRB- uses recursion to search for both exact and approximate tandem repeats . Repeating unit lengths of 2 to 5 are sought , and a score is used to determine location . Tandem Repeat Occurrence Locator -LRB- TROLL -RRB- -LSB- 8 -RSB- , uses a keyword tree adapted from bibliographic searching techniques and attempts to match the keywords exactly but it does not specify the positions of repeats . In this work , we describe a program called SSRscanner -LRB- Simple sequence repeat scanner -RRB- that uses dictionary approach to find simple sequence repeats of pre-selected motifs . SSRscanner is a PERL script developed for scanning genomes to find repeats of any length , their exact position on chromosome and frequency of occurrence . It is fast and requires a standard Personal Computer -LRB- PC -RRB- and PERL to operate . SSRscanner can accept large sequences as input and large number of motifs can be searched simultaneously . Thus , the running time of the program is greatly reduced . To demonstrate the use of SSRscanner , Arabidopsis thaliana genome was analyzed for finding out distribution , frequency and specific position of SSRs in the genome . -LSB- 9 -RSB- The advantages over many other programs developed for SSR identification includes its ability to search motifs of any length repeated for a number of times and to give the exact position of the motif in the genome . Methodology Program Input SSRscanner -LRB- implemented in PERL -RRB- accepts two text files -LRB- . txt -RRB- . Upon execution of the program , it prompts to enter the file name containing the DNA sequence data . It also prompts for a file containing motifs of different repeat types . It then prompts for the number of times for the motifs to be repeated -LRB- for example for searching tri-nucleotide repeats the user should enter 3 -RRB- -LRB- Figure 1B -RRB- . Input sequence and motifs are parsed to SSRscanner that extracts the SSRs giving their distribution/frequency and specific location . The results from SSRscanner are appended into result files -LRB- Figure 1A -RRB- . Program output SSRscanner gives two files in output -LRB- Figure 1A -RRB- . They are -LRB- 1 -RRB- Motifposition.txt -LRB- gives the frequency of each repeat provided in the motif file -RRB- and -LRB- 2 -RRB- Motifresult.exe -LRB- gives the specific location of each repeat -RRB- . The data obtained can then be arranged into desired formats . Caveats and Future Development SSRscanner is a PERL script and it requires PERL to be installed on a PC before running the program . We are developing a web based CGI for SSRscanner .

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scanner ssr repeats script microsatellite