Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus WK1
- Jimmy H Saw†1, 10,
- Bruce W Mountain†2,
- Lu Feng†3, 4, 5,
- Marina V Omelchenko†6,
- Shaobin Hou†7,
- Jennifer A Saito1,
- Matthew B Stott2,
- Dan Li3, 4, 5,
- Guang Zhao3, 4, 5,
- Junli Wu3, 4, 5,
- Michael Y Galperin6,
- Eugene V Koonin6,
- Kira S Makarova6,
- Yuri I Wolf6,
- Daniel J Rigden8,
- Peter F Dunfield9,
- Lei Wang3, 4, 5Email author and
- Maqsudul Alam1, 7Email author
© Saw et al.; licensee BioMed Central Ltd. 2008
Received: 12 June 2008
Accepted: 17 November 2008
Published: 17 November 2008
Gram-positive bacteria of the genus Anoxybacillus have been found in diverse thermophilic habitats, such as geothermal hot springs and manure, and in processed foods such as gelatin and milk powder. Anoxybacillus flavithermus is a facultatively anaerobic bacterium found in super-saturated silica solutions and in opaline silica sinter. The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life.
We report here the complete genome sequence of A. flavithermus strain WK1, isolated from the waste water drain at the Wairakei geothermal power station in New Zealand. It consists of a single chromosome of 2,846,746 base pairs and is predicted to encode 2,863 proteins. In silico genome analysis identified several enzymes that could be involved in silica adaptation and biofilm formation, and their predicted functions were experimentally validated in vitro. Proteomic analysis confirmed the regulation of biofilm-related proteins and crucial enzymes for the synthesis of long-chain polyamines as constituents of silica nanospheres.
Microbial fossils preserved in silica and silica sinters are excellent objects for studying ancient life, a new paleobiological frontier. An integrated analysis of the A. flavithermus genome and proteome provides the first glimpse of metabolic adaptation during silicification and sinter formation. Comparative genome analysis suggests an extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli.
Gram-positive bacteria of the genus Anoxybacillus were originally described as obligately anaerobic spore-forming bacilli. They are members of the family Bacillaceae, whose representatives were long believed to be obligate or facultative aerobes. However, it has been shown that Bacillus subtilis and several other bacilli are capable of anaerobic growth [1–3], whereas Anoxybacillus spp. turned out to be facultative anaerobes [4, 5]. They are found in diverse moderate- to high-temperature habitats such as geothermal hot springs, manure, and processed foods such as gelatin [4, 6, 7]. Anoxybacillus flavithermus is a major contaminant of milk powder .
We report here the complete genome sequence of the thermophilic bacterium A. flavithermus strain WK1 [GenBank:CP000922], which was isolated from the waste water drain at the Wairakei geothermal power station in New Zealand . This isolate has been deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as strain DSM 21510. The 16S rRNA sequence of strain WK1 is 99.8% identical to that of the A. flavithermus type strain DSM 2641 , originally isolated from a hot spring in New Zealand . The name 'flavithermus' reflects the dark yellow color of its colonies, caused by accumulation of a carotenoid pigment in the cell membrane. Anoxybacillus flavithermus, formerly referred to as 'Bacillus flavothermus', grows in an unusually wide range of temperatures, 30-72°C, and pH values, from 5.5 to 10.0 . Temperature adaptation mechanisms in A. flavithermus proteins have attracted some attention to this organism . However, a property of greater potential importance to the fields of paleobiology and astrobiology is its ability to grow in waters that are super-saturated with amorphous silica, and where opaline silica sinter is actively forming [9, 12]. Flushed waste geothermal fluids from the Wairakei power station drain into a concrete channel at about 95°C. These fluids cool as they travel down the 2-km-long drainage channel, dropping to 55°C before entering Wairakei Stream. As the water cools down, silica sinter deposits subaqueously in the channels, forming precipitates composed of amorphous silica (opal-A) . The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life . Although bacteria are believed to play only a passive role in silicification, they definitely affect the absolute rate of silica precipitation by providing increased surface area. In addition, bacteria largely control the textural features of the resulting siliceous sinters . We have obtained the complete genome sequence of A. flavithermus WK1 and employed it to analyze bacterial physiology and its changes in response to silica-rich conditions. This study sheds light on the biogeochemical processes that occur during the interaction between microbial cells and dissolved silica and result in sinter deposition.
Genome features of A. flavithermus
Number of predicted coding sequences
2,863, 104 RNA, 112 pseudogenes
Average size of coding sequences
Number of protein coding genes
2,863 (22 with frame shifts)
Number of proteins with assigned biological function
Number of proteins with predicted general function
Number of proteins of unknown function
Number of proteins assigned to COGs
Number of tRNA genes
Number of rRNA operons
Number of small RNA genes
Number of riboswitches
Despite its much smaller genome size, A. flavithermus appears to retain most of the key metabolic pathways present in B. subtilis and other bacilli. It has a complete set of enzymes for biosynthesis of all amino acids, nucleotides and cofactors, with the sole exception of the molybdenum cofactor (Table S2 in Additional data file 1). Cells of A. flavithermus had been originally reported to reduce nitrate [4, 6]; however, in subsequent work, nitrate reductase activity has not been observed in this organism . In accord with the latter report, the A. flavithermus WK1 genome encodes neither the assimilatory nitrate/nitrite reductase complex (NasBCDE) nor the respiratory nitrate reductase complex (NarGHJI), both of which are present and functional in B. subtilis [16, 17], nor the third (proteobacterial) type of nitrate reductase (NapAB) . Nitrate/nitrite transporters NasA and NarK are missing in A. flavithermus as well. The loss of nitrate reductases in A. flavithermus WK1 appears to be a recent event, given that G. kaustophilus encodes the assimilatory nitrate reductase, whereas G. thermodenitrificans encodes the respiratory nitrate reductase complex. In accordance with the loss of nitrate reductases, A. flavithermus WK1 has lost the entire set of enzymes involved in the biosynthesis of the molybdenum cofactor of nitrate reductase, as well as the molybdate-specific ABC (ATP-binding cassette)-type transporter, all of which are encoded in G. kaustophilus and G. thermodenitrificans. Molybdenum-dependent xanthine dehydrogenase and its homologs YoaE (putative formate dehydrogenase) and YyaE have been lost as well. As suggested in , the loss of molybdate metabolism could be part of a strategy to avoid generation of reactive oxygen species.
Electron transport and oxygen resistance genes of A. flavithermus
B. subtilis orthologs
Cytochrome bd-type quinol oxidase
Cytochrome aa3-type quinol oxidase
Electron transfer flavoprotein
Menaquinol:cytochrome c oxidoreductase
Cytochrome c oxidase (caa3-type)
Response to oxygen
Catalase (peroxidase I)
Redox sensing and cytochrome biogenesis system
Anoxybacillus flavithermus WK1 grows well anaerobically in rich media, such as tryptic soy broth (TSB). Owing to the absence of nitrate and nitrite reductases (see above), its anaerobic growth cannot rely on nitrate or nitrite respiration and apparently proceeds by fermentation. Fermentative growth of B. subtilis requires phosphotransacetylase, acetate kinase and L-lactate dehydrogenase genes [1, 3]. All these genes are conserved in A. flavithermus (pta, Aflv_2760; ack, Aflv_0480; lctE, Aflv_0889), suggesting that, like B. subtilis, this bacterium can ferment glucose and pyruvate into acetate . However, catabolic acetolactate synthase AlsSD and acetolactate dehydrogenase, which are responsible for acetoin production by fermenting B. subtilis , are missing in A. flavithermus, indicating that it cannot produce acetoin.
In agreement with the experimental data , genome analysis indicates that A. flavithermus is able to utilize a variety of carbohydrates as sole carbon sources. It has at least four sugar phosphotransferase systems with predicted specificity for glucose, fructose, sucrose, and mannitol. Additionally, it encodes ABC-type transporters for ribose, glycerol-3-phosphate, and maltose, and several ABC-type sugar transporters of unknown specificity. A complete set of enzymes was identified for general carbohydrate metabolism (glycolysis, the TCA cycle, and the pentose phosphate pathway, but not the Entner-Doudoroff pathway). The A. flavithermus genome also contains a gene cluster (Aflv_2610-2618) that is very similar to the gene cluster associated with antibiotic production and secretion in many other Gram-positive bacteria , suggesting that A. flavithermus might be able to produce bactericidal peptides. It is not obvious which of these systems are relevant to the survival of A. flavithermus in silica solutions, but they might facilitate its growth in powdered milk and similar habitats.
Evolution of the Anoxybacillusbranch of bacilli
Being a free-living environmental microorganism, A. flavithermus encodes numerous proteins involved in signal transduction. These include 23 sensor histidine kinases and 24 response regulators (16 pairs of which are clustered in operons), 20 methyl-accepting chemotaxis proteins, 5 predicted eukaryotic-type Ser/Thr protein kinases, and 21 proteins involved in metabolism of cyclic diguanylate (cyclic (3',5')-dimeric guanosine monophosphate (c-di-GMP)), a recently recognized secondary messenger that regulates transition from motility to sessility and biofilm formation in a variety of bacteria . Compared to other bacilli, this set is significantly enriched in chemotaxis transducers and c-di-GMP-related proteins . Anoxybacillus flavithermus encodes 12 proteins with the diguanylate cyclase (GGDEF) domain, 6 of which also contain the c-di-GMP phosphodiesterase (EAL) domain, and one combines GGDEF with an alternative c-di-GMP phosphodiesterase (HD-GYP) domain. Anoxybacillus flavithermus WK1 also encodes two proteins with the EAL domain and seven proteins with the HD-GYP domain that do not contain the GGDEF domain. In addition, it encodes two proteins with the PilZ domain , which serves as a c-di-GMP-binding adaptor protein [28, 29]. The total number of proteins implicated in c-di-GMP turnover in A. flavithermus is third highest among all Gram-positive bacteria sequenced to date, after Clostridium difficile and Desulfitobacterium hafniense, which have much larger genomes [26, 30].
Silicification of A. flavithermuscells and biofilm formation
A. flavithermus orthologs of biofilm-related genes of B. subtilis
Biofilm formation protein
ABC transporter subunit
Regulatory protein (regulator of ComK)
Camelysin, spore coat-associated metalloprotease
Capsular polysaccharide biosynthesis protein EpsG
Capsular polysaccharide biosynthesis glycosyl transferase EpsH
Cell adaptation to silica
We also examined protein expression profiles in the cells grown in the presence or absence of silica for 7 days. Sinters started forming in the silica-containing sample 5 days after inoculation, so by the end of the incubation the cells became silicified. Owing to the problems with collecting and analyzing silicified A. flavithermus cells, no attempt has been made to replicate this experiment, so these results were only considered in comparison to the samples from 8-hour exposure to silica. Spermine synthase Aflv_1437 was not detected in either silicified or control cells (last column of Table S4 in Additional data file 1), and arginase (Aflv_0146; Figure S7 in Additional data file 1) was only detected in the silicified cells at very low abundance. In contrast, spermidine synthase Aflv_2750 was detected at similar levels in both types of cells, indicating general cellular functions for spermidine. Remarkably, the transcriptional regulator AbrB (Aflv_0031) remained moderately up-regulated in the silicified cells, suggesting that it might play a general role in silica adaptation of A. flavithermus. Also up-regulated in both silica conditions were chemotaxis response regulator CheY (Aflv_1727), thiol peroxidase Aflv_0478, which is apparently involved in antioxidant defense, and methionine aminopeptidase Aflv_0127. Those proteins could also play a role in silica niche adaptation of A. flavithermus.
Silica precipitation and formation of sinter is an important geochemical process in hot spring systems, and understanding how these structures form might be important for deciphering some of the earliest biological processes on Earth [13, 14].
Microbial fossils are well preserved in silica compared to CaCO3 or iron precipitates , and silica sinters are excellent structures for studying ancient microbial life. Microorganisms were previously believed to play no active role in the formation of silica precipitates. Rather, microbial cell surfaces have been assumed to provide nucleation sites to allow precipitation of minerals . However, several recent studies have shed light on the biotic components that might play an active role in silicification. The best studied in this respect are diatoms, which build silica nanostructures in a controlled manner and under ambient conditions [44, 45]. Formation of silica nanostructures in diatoms is influenced by polycationic peptides, named silaffins [39, 46], and LCPAs . In diatom cells, silica is deposited as nanospheres before being transformed into complex structures [48, 49]. Polyamines have been shown to catalyze siloxane-bond formation and can also act as flocculating agents, leading to silica polymerization [50, 51]. In the bacterial world, polyamines have been shown to be essential for biofilm formation in Yersinia pestis  and to activate biofilm formation in Vibrio cholerae, although, in the latter case, the effect appeared to be due primarily to intracellular signaling . Studies of silicate binding by B. subtilis cell walls by Terry Beveridge and colleagues showed that it was electrostatic in nature and depended on the surface charge [54, 55]. The observations of silica nanospheres formed around the bacterial cells in hot springs  and in simulated experimental conditions with A. flavithermus (Figure 5e) suggest that silica formation in hot springs also might be biologically influenced.
LCPAs participate in silica formation in diatoms [40–42] and enzymes similar to spermidine and spermine synthases are thought to be required for their synthesis . On the other hand, polyamines, including putrescine, spermidine, and spermine, are ubiquitous in all cells, and play essential roles in cell proliferation and differentiation [57, 58]. Of the two speE paralogs in A. flavithermus WK1, SpeE (Aflv_2750) catalyzes the formation of spermidine from putrescine, most likely for general cellular functions, whereas the SpeE-like Aflv_1437 catalyzes the conversion of putrescine into spermine and could be an important part of LCPA production. In B. subtilis, polyamines are synthesized via a single route, the agmatine pathway encoded by speA and the speEB operon . Enzymes for this route are also encoded in A. flavithermus and most likely serve normal cellular functions as the expression level of arginine decarboxylase (Aflv_1886), the key enzyme of the pathway, was not stimulated by silica. Therefore, up-regulation of putrescine production for SpeE-like production was through the other route catalyzed by arginase and ornithine decarboxylase. The presence of two putrescine synthesis routes and two putrescine aminopropyltransferase homologs (SpeE and SpeE-like) indicates that polyamine synthesis is crucial for the specific niche adaptation of A. flavithermus.
Based on the proposed LCPA synthesis pathway (Figure 6), conversion of putrescine into spermine by the SpeE-like protein Aflv_1437 could be followed by further transfer of aminopropyl groups leading to the formation of LCPAs. Previous studies using computer simulations have shown that polyamine chains may self-assemble into structures serving as scaffolding or nucleation sites for the precipitation of silica-polyamine complexes . Our results suggest that the SpeE-like enzyme may be responsible for the production of LCPAs that form the basis or scaffolding needed for the silica-polyamine complexes to aggregate.
Biofilm formation and production of exopolysaccharides are important processes that could facilitate silica sinter formation in hot springs. The abundance of c-di-GMP-related proteins in the A. flavithermus genome, as well as the up-regulation of the global regulator AbrB (Aflv_0031) in the presence of silica, suggests that biofilm formation by this organism is part of its global response to silica. In studies of the cyanobacterium Calothrix sp., silicification had no significant effect on cell viability ; there is little doubt that A. flavithermus cells remain viable during silicification as well. Our current working model implies that polymerization of monomeric and polymeric silica into silica nanospheres is facilitated by biotic factors such as LCPAs, as indicated by our proteomics results. Attachment of these silica nanospheres to the exopolysaccharide coating surrounding the A. flavithermus cells (Figure 5e) is a key step in silica sinter formation. In summary, this integrated genomics and proteomics study provides the first experimental evidence of the biochemical reactions between dissolved silica and the bacterial cell. Such reactions are likely to be crucial in the preservation of ancient microbial life and the growth of modern hot spring sinter deposits.
The complete genome sequence of A. flavithermus shows clear signs of genome compaction in the Anoxybacilus/Geobacillus branch, compared to other members of the family Bacillaceae. In A. flavithermus strain WK1, adaptations to growth at high temperatures in supersaturated silica solutions include general streamlining of the genome, coupled with preservation of the major metabolic pathways and the capability to form biofilms. The presence of bacteria appears to affect silicification in several different ways. Passive effects of bacteria include providing nucleation sites for sinter formation and an increased surface area for silica precipitation. In addition, synthesis of LCPAs and biofilm formation by A. flavithermus could regulate sinter formation and control the textural features of the resulting siliceous sinters. The presence of an array of c-di-GMP-related signal transduction proteins suggests that A. flavithermus could regulate biofilm formation in response to the environmental conditions.
Materials and methods
Sequencing, assembly, and annotation
The genome of A. flavithermus was sequenced using the whole-genome-shotgun approach as previously described , using genomic DNA that was randomly sheared to generate 3 kb and 6 kb fragments. These fragments were size-selected on agarose gels, purified, end-repaired, ligated to pUC118 vectors, and transformed into DH10B competent cells by electroporation. Plasmids from positive clones were sequenced using Beckman CEQ 8000 (Beckman Coulter, Fullerton, CA, USA) and ABI 3730xl (Applied Biosystems, Foster City, CA, USA) sequencers. A total of 55,975 valid sequences were used for assembly with PHRED/PHRAP/CONSED , CAP3 , and SEQMAN II (DNAStar) programs. Further 3,863 sequences were used to close gaps between contigs and to improve overall sequence quality of contigs. Long PCR reactions were performed to verify sequence assembly. Protein-coding genes were predicted using GLIMMER  followed by BLASTX  searches of intergenic regions between predicted ORFs. Transfer RNAs were predicted by tRNAscan-SE . Genome annotation was performed by running BLAST and PSI-BLAST against the NCBI protein database and the COG database with manual verification as described previously . Metabolic pathways were analyzed by comparing COG assignments of A. flavithermus proteins with the standard sets of COGs involved in each pathway . Phylogenetic analysis was performed as described .
Biofilm formation and silica precipitation
Biofilm formation by A. flavithermus cells grown in the presence of silica was studied by incubating the cells in a chemostat-like system, consisting of a 500 ml serum vial, capped with a rubber seal with two input and one output lines. This was filled halfway with 300 ml of TSB. The two input lines were fed through the rubber seal and connected via peristaltic pumps to sterile reservoirs. One reservoir contained 2 × TSB and the other water. They were each fed at 0.15 ml per minute giving 1 × TSB in the vial. An output line connected to another peristaltic pump maintained the medium level at 300 ml. A final output line was fit with a luer valve and syringe to allow samples to be removed from the reservoir. A glass slide stood upright in the vial as a substrate for silica sinters, to be observed by scanning electron microscopy at the conclusion of the experiment. The cultivation vessel was contained in a 60°C oven and shaken gently at 100 rpm to simulate wave motion. After running the system for two days to ensure it was sterile, the medium was inoculated with A. flavithermus WK1 through the luer-fitted line. The system ran for two days to build up cell mass, then samples of 200 ml were taken for three successive days. Samples were centrifuged, the pellet washed 3 times in buffer (68 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 9 mM NaH2PO4, 50 mM TRIS, pH 8.0), stored at -20°C and later freeze-dried. The system was running at pH 5.8 and OD600 0.15 during this time. After three days, the water was replaced with 1,000 mg/kg silica solution adjusted to pH 7. This flowed through a 200°C oven before reaching the cultivation vessel in order to monomerize the silica and sterilize the water. Samples were taken after 1, 2, 3 and 7 days and prepared as above. As time progressed, there was increasingly more solid, amorphous silica in the vessel, as this was not removed by the outflow. The system remained at pH 5.8 but there was no longer any way to reliably measure OD600 because of the silica precipitate. At the end of incubation, some of the amorphous silica and the slide were removed to be fixed in 2% glutaraldehyde. Samples for scanning electron microscopy were removed from storage and allowed to air-dry before coating with gold/palladium. Scanning electron microscopy examination was done on a Hitachi S-800 Field Emission scanning electron microscope operating at 15 kV.
Anoxybacillus flavithermus cells were grown in TSB at 60°C with shaking at 200 rpm on an orbital shaker (Thermo Electron Co., Waltham, MA, USA) to OD600 of 0.6, followed by the addition of silica to a final concentration of 10.7 mM and growth for another 8 hours. The same batch of the culture without added silica served as the control. Cells were harvested by centrifugation at 10,000 × g at 4°C for 10 minutes, extracellular proteins from the supernatant were collected and cellular proteins from the pellet were solubilized . Immunoelectrophoresis (the first dimension) was carried out on IPG strips (Amersham Pharmacia Biotech, Uppsala, Sweden) in a Multiphor II electrophoresis unit (Amersham Pharmacia Biotech) with running conditions as described by Büttner et al. . For the second dimension, vertical slab SDS-PAGE (12%) was run in a Bio-Rad Protean II Xi unit (Bio-Rad Laboratories, Hercules, CA, USA). Gels were stained with colloidal CBB G-250 , and scanned with a PowerLook 1000 (UMAX Technologies Inc., Fremont, CA, USA). PDQuest version 7.3.0 (Bio-Rad) was used for image analysis. Proteins were classified as being differentially expressed under the two conditions when spot intensity showed at least 1.5-fold change.
For protein identification, spots were excised from the gels, washed with 25 mM NH4HCO3 in 50% (v/v) acetonitrile for 3 × 15 minutes at room temperature, dried in a vacuum centrifuge, and incubated in 50 μl digestion solution consisted of 25 mM NH4HCO3 in 0.1% acetic acid and 12.5 ng/mL of trypsin (Promega, Madison, WI, USA) at 37°C overnight. The digested protein (0.3 μl) was spotted on a MALDI sample plate with the same volume of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid). Peptide mass spectra were obtained on a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems) in the positive ion reflector mode. The mass spectrometry spectra were internally calibrated with a mass standard kit for the 4700 Proteomics Analyzer. Proteins were identified by automated peptide mass fingerprinting using the Global Proteome Server Explorer™ software (Version 3.5, Applied Biosystems) against an in-house sequence database of A. flavithermus proteins. Peak lists (S/N > 10) were extracted from raw data for the data processing, and positive identifications were accepted up to 95% of confidence level. The following criteria were used for the database searches: maximum one missed cleavage per peptide; mass tolerance of 0.1 Da, and the acceptation of carbamidomethylation for cysteine and oxidation for methionine.
Characterization of enzymes involved in LCPA synthesis
The genes Aflv_0024, Aflv_1886, Aflv_2749, and Aflv_1437 were cloned into the pET-14b vector, Aflv_0146 into pET-3a, and Aflv_2750 into pET-28a. Escherichia coli strain BL21 carrying each of the recombinant plasmids was grown overnight with shaking at 37°C in Luria broth containing 100 mg/ml ampicillin. The overnight culture (4 ml) was inoculated into 400 ml of fresh Luria broth and grown to mid-log phase (A600 = 0.6). Expression of Aflv_0146, Aflv_1437, Aflv_1886, Aflv_2749, and Aflv_2750 products was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 4 hours, and expression of the Aflv_0024 product was induced with 0.1 mM IPTG at 12°C for 8 hours. After IPTG induction, the cells were harvested by centrifugation at 6,000 × g at 4°C for 5 minutes, washed with binding buffer (10 mM imidazole, 300 mM NaCl and 50 mM Tris-HCl, pH 8.0), resuspended in 5 ml of binding buffer containing 1 mM phenylmethylsulfonyl fluoride and 1 mg/ml of lysozyme, and sonicated for 10 1-minute cycles with 1-second pulse on alternating 1-second pulse off at 95% of the maximum power (200 W) using an UP200S Ultraschallprozessor with a tapered microtip. The lysate of Aflv_1886 was further incubated for 10 minutes at 60°C. After centrifugation at 12,000 × g at 4°C for 30 minutes, the crude extract containing 6× His-tagged fusion proteins was purified by nickel ion affinity chromatography with a Chelating Sepharose Fast Flow column (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions. The column was washed successively with 100 ml of wash buffer (25 mM imidazole, 300 mM NaCl, and 50 mM Tris-HCl, pH 8.0), and the fusion proteins were eluted with the elution buffer (250 mM imidazole, 300 mM NaCl and 50 mM Tris-HCl, pH 8.0), and dialyzed in 0.1 M Tris-HCl buffer (pH 8.8). Protein concentration was determined by the Bradford method. For SDS-PAGE, proteins were denatured at 100°C for 5 minutes in the presence of 0.1% SDS and 1% 2-mercaptoethanol, loaded in a 5% (w/v) stacking gel and separated in a 10% (w/v) separation gel. The gel was stained with Coomassie Bright Blue R250. The molecular weight markers were from the LMW-SDS Marker Kit (GE Healthcare).
Reactions catalyzed by arginase, arginine decarboxylase, ornithine decarboxylase, agmatinase, spermidine synthase and spermine synthase were carried out as previously described [71–74]. The activities of arginase and arginine decarboxylase were determined by thin-layer chromatography . The activities of the other enzymes were assayed by high-performance liquid chromatography (HPLC) after Schotten-Baumann benzoylation as previously described [74, 76, 77]. HPLC analysis was performed with a Venusil XBP C18 column (4.6 × 250 mm) in conjunction with a LC-20AT (Shimadzu, Kyoto, Japan) HPLC apparatus. Benzoyl putrescine, spermidine and spermine were eluted by a gradient started with 60% methanol in water and proceeded linearly to 100% methanol, with a flow rate of 0.8 ml/minute over 20 minutes, and detected at a wavelength of 229 nm.
Additional data files
The following additional data are available. Additional data file 1 contains Figures S1-S7 and Tables S1-S4.
cyclic (3',5')-dimeric guanosine monophosphate
clusters of orthologous groups of proteins
high-performance liquid chromatography
matrix-assisted laser desorption/ionization-time of flight
open reading frame
tryptic soy broth.
This study was supported by the University of Hawaii and US DoD W81XWH0520013 and Maui High Performance Computing Center to MA, and by the Intramural Research Program of the National Library of Medicine at the National Institutes of Health (MVO, MYG, EVK, KSM and YIW). This study is dedicated to the memory of Dr Terry Beveridge, a pioneer in studies of bacterial surfaces.
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