Growth of Hyperthermophilic Archaeon
            <i>Pyrococcus furiosus</i>
            on Chitin Involves Two Family 18 Chitinases

Gao, Jun; Bauer, Michael; Shockley, Keith R.; Pysz, Marybeth A.; Kelly, Robert M.

doi:10.1128/aem.69.6.3119-3128.2003

Cited by 80 publications

(59 citation statements)

References 78 publications

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“…Maltose, laminarin, cellobiose, starch, and chitin were obtained from Sigma (St. Louis, MO), and barley glucan was obtained from Megazyme (Wicklow, Ireland). Growth on chitin was performed as described previously (16). Each glucan was added to the medium at a final concentration of 3.3 g/liter prior to inoculation.…”

Section: Methodsmentioning

confidence: 99%

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Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

et al. 2006

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Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these ␣-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-␣-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of ␣-glucan substrates by P. furiosus.Pyrococcus furiosus is an obligately anaerobic, heterotrophic, hyperthermophilic archaeon that was isolated from a shallow hydrothermal vent near Vulcano Island, Italy (12). The organism grows optimally near 100°C and is able to utilize a range of sugars as a primary carbon source (4,11,28,29). These include cellobiose (28, 43), laminarin (43), chitin (16), maltose (29), barley glucan (3), and starch (29). However, the means by which these compounds are metabolized by P. furiosus are not well understood. In attempting to define the pathway by which starch, and one of its degradation products, maltose, serve as carbon sources, there are two complicating factors. First, the genome sequence of P. furiosus (36) contains an array of genes that encode putative glycosyl hydrolases and glycosyl transferases. With respect to starch, they include five enzymes that potentially hydrolyze ␣-glucans. Two of them (PF0477 and PF1935) have putative signal peptides and are presumably extracellular, while the other three (PF0272, PF0478, and PF1939) lack a signal peptide and are presumably intracellular. They are annotated as amylases (PF0272 and PF0477), amylopullulanases (PF1934 and PF1935), and cyclodextrin glucanotransferases (PF0478). The question is, are all of these enzymes involved in degrading starch? The pathway for utilizing maltose and maltodextrins, primary products of starch breakdown, is well defined in ...

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Section: Methodsmentioning

confidence: 99%

“…The organism grows optimally near 100°C and is able to utilize a range of sugars as a primary carbon source (4,11,28,29). These include cellobiose (28,43), laminarin (43), chitin (16), maltose (29), barley glucan (3), and starch (29). However, the means by which these compounds are metabolized by P. furiosus are not well understood.…”

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confidence: 99%

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

et al. 2006

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“…To ensure minimum carryover between substrates, cells were grown for at least six passes on each carbon source using a 0.5% (vol/vol) starting inoculum before obtaining the growth curves. Isolation of total RNA from T. maritima was performed on cells that were grown until early-to mid-exponential phase on the various growth substrates, using a protocol described previously (18).…”

Section: Methodsmentioning

confidence: 99%

An Expression-Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in theHyperthermophilic BacteriumThermotoga maritima

et al. 2005

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Comprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixedmodel analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T. maritima relies extensively on ABC transporters for carbohydrate uptake, many of which are likely controlled by local regulators responsive to either the transport substrate or a key metabolic degradation product. Roles in uptake of specific carbohydrates were suggested for members of the expanded Opp/Dpp family of ABC transporters. In this family, phylogenetic relationships among transport systems revealed patterns of possible duplication and divergence as a strategy for the evolution of new uptake capabilities. The presence of GC-rich hairpin sequences between substrate-binding proteins and other components of Opp/Dpp family transporters offers a possible explanation for differential regulation of transporter subunit genes. Numerous improvements to T. maritima genome annotations were proposed, including the identification of ABC transport systems originally annotated as oligopeptide transporters as candidate transporters for rhamnose, xylose, ␤-xylan, and ␤-glucans and identification of genes likely to encode proteins missing from current annotations of the pentose phosphate pathway. Beyond the information obtained for T. maritima, the present study illustrates how expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable.Thermotoga maritima, a hyperthermophilic anaerobe with an optimal growth temperature of 80°C, has been found in diverse high-temperature locations and is capable of using a wide variety of simple and complex carbohydrate substrates for growth. The complexity of its carbohydrate utilization strategies, revealed by genome sequencing (48) and through previous work (11,12,47,51), is surprising, given the primitive features of this microorganism. Considerable genomic plasticity has been observed even within the Thermotoga genus, with respect to the gene content of carbohydrate active enzymes and transporter subunits, which may to some extent relate to lateral gene transfer events (48,49). Despite the range of sugar-active enzymes found within T. maritima MSB8 genome (Table S1 in the supplemental material) (6,9,10,23,27,34,35,37,38,39,42,45,46,54,70,71), a PTS (phosphoenolpyruvatedependent phosphotransferase system) similar to those used by other species for preferential uptake of selected sugars is apparently absent (48). No homologs of the PTS components EI and HPr (phosphocarrier proteins) and no sugar-specific EII sugar transporter subunits have been identified ...

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“…For RNA sampling, 350 ml of each culture was harvested into centrifuge bottles and cooled rapidly to 0°C by immersion in ice water, followed by immediate centrifugation of the cells at 10,000 ϫ g for 15 min at 4°C. RNA extraction was then performed following established protocols (7). RNA/cDNA obtained previously during syntrophic coculture of T. maritima with M. jannaschii (11) was reexamined.…”

Section: Methodsmentioning

confidence: 99%

Colocation of Genes Encoding a tRNA-mRNA Hybrid and a Putative Signaling Peptide on Complementary Strands in the Genome of the Hyperthermophilic BacteriumThermotoga maritima

et al. 2006

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In the genome of the hyperthermophilic bacterium Thermotoga maritima, TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA, a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans-translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii. TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.Although canonical models for bacterial signaling mechanisms have been established (4, 9), chemically diverse molecules that act as mediators for cell-to-cell communication continue to be discovered (42), concomitant with the expanding range of microorganisms found to be involved in inter-and intraspecies communication (18,20,36,42). Recently, signaling has been connected with biologically extreme environments. For example, N-acyl homoserine lactones have been reported in cultures of the haloalkaliphilic archaeon Natronococcus occultus (28). Furthermore, cell density-dependent, peptide-based signaling, tied to formation of exopolysaccharide-based cell aggregates, was identified in the hyperthermophilic bacterium Thermotoga maritima in synthrophic coculture with the hyperthermophilic archaeon Methanococcus jannaschii (10, 11). Presumably, the formation of such aggregates allows M. jannaschii to make methane using the H 2 produced by T. maritima cells as an auto-inhibitory by-product of carbohydrate metabolism (22). During syntrophic coculture of these two organisms, examination of genome-wide transcriptional profiles indicated increased transcript levels of a small hypothetical open reading frame (ORF), annotated as TM0504, in the T. maritima genome (25), in conjunction with up-regulation of adjacent ORFs annotated as putative ABC transporter permease and ATPase subunits (11). Subsequent reexamination of the genomic neighborhood of the TM0504 gene revealed that it was colocated on the complementary strand within the ssrA gene encoding a highly structure...

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Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases

Cited by 80 publications

References 78 publications

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

An Expression-Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in theHyperthermophilic BacteriumThermotoga maritima

Colocation of Genes Encoding a tRNA-mRNA Hybrid and a Putative Signaling Peptide on Complementary Strands in the Genome of the Hyperthermophilic BacteriumThermotoga maritima

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