Anaerobic coculture of microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C enhances generation of n-alkane-rich biofuels after pyrolysis.

We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH(4) production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

Diversity and similarity of microbial communities in petroleum crude oils produced in Asia.

To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

Bacillus minimum genome factory: effective utilization of microbial genome information.

In 1997, the complete genomic DNA sequence of Bacillus subtilis (4.2 Mbp) was determined and 4100 genes were identified [Kunst, Ogasawara, Moszer, Albertini, Alloni, Azevedo, Bertero, Bessieres, Bolotin, Borchert, S. et al. (1997) Nature 90, 249-256]. In addition, B. subtilis, which shows an excellent ability to secrete proteins (enzymes) and antibiotics in large quantities outside the cell, plays an important role in industrial and medical fields. It is necessary to clarify the genes involved in the production of compounds by understanding the network of these 4100 genes and the proceeding analysis of genes of unknown functions. In promoting such a study, it is expected that the regulatory system of B. subtilis can be simplified by the creation of a Bacillus strain with a reduced genome by discriminating genes unnecessary for the production of proteins from essential genes, and deleting as many of these unnecessary genes as possible, which may help to understand this complex network of genes. We have previously distinguished essential and non-essential genes by evaluating the growth and enzyme-producing properties of strains of B. subtilis in which about 3000 genes (except 271 essential genes) have been disrupted or deleted singly, and have successfully utilized the findings from these studies in creating the MG1M strain with an approx. 1 Mbp deletion by serially deleting 17 unnecessary regions from the genome. This strain showed slightly reduced growth in enzyme-production medium, but no marked morphological changes. Moreover, we confirmed that the MG1M strain had cellulase and protease productivity comparable with that of the B. subtilis 168 strain, thus demonstrating that genome reduction does not contribute to a negative influence on enzyme productivity.

Bacillus subtilis AprX involved in degradation of a heterologous protein during the late stationary growth phase.

In Bacillus subtilis, extracellular protease-deficient mutants have been used in attempts to increase the productivity of heterologous proteins. We detected protease activity of AprX using protease zymography in the culture medium at the late stationary growth phase. An alpha-amylase-A522-PreS2 hybrid protein, in which the PreS2 antigen of human hepatitis B virus (HBV) is fused with the N-terminal 522-amino-acid polypeptide of B. subtilis alpha-amylase, has been produced in multiple-protease-deficient mutants. The B. subtilis KA8AX strain, which is deficient in eight extracellular proteases and AprX, did not show the proteolysis of alpha-amylase-A522-PreS2 in the late stationary growth phase. Moreover, the production of alpha-amylase-A522-PreS2 was about 80 mg/l, which was eight times higher than that by the KA8AX strain previously reported. In addition, we showed the degradation of the heterologous protein by AprX that leaked to the culture medium (probably caused by cell lysis) during the late stationary growth phase.

Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis.

The Bacillus subtilis spo0A mutant is an adequate host for extracellular protein production (e.g., alpha-amylase). However the mutant was prone to cell lysis. SDS-PAGE and zymography of cell wall lytic proteins indicated that the spo0A mutant contained high amounts of two major autolysins (LytC [CwlB] and LytD [CwlG]) and two minor cell wall lytic enzymes (LytE [CwlF] and LytF [CwlE]). On the other hand, the expression of eight extracellular protease genes was very poor or absent in the spo0A mutant. An eight-extracellular-protease-deficient mutant (Dpr8 strain) was constructed and the strain also exhibited cell lysis. The autolysins from the spo0A mutant were degraded by the supernatant of the wild type but not degraded by that of the Dpr8 mutant. These results suggest that the extensive cell lysis of the spo0A mutant was partially caused by the stability of autolysins via the decrease of the extracellular proteases. The introduction of a major autolysin and/or SigD mutations into the spo0A mutant was effective for preventing cell lysis.

Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707.

Maltohexaose-producing amylase (G6-amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short-chain amylose (DP=17). Our previous crystallographic study showed that G6-amylase has nine subsites, from -6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6-amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6-amylase complexes with G6 and maltopentaose (G5). In the active site of the G6-amylase/G5 complex, G5 is bound to subsites -6 to -2, while G1 and G6 are found at subsites +2 and -7 to -2, respectively, in the G6-amylase/G6 complex. In both structures, the glucosyl residue located at subsite -6 is stacked to the indole moiety of Trp140 within a distance of 4A. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short-chain amylose by W140L is lower than that by W140Y or wild-type enzyme. The face-to-face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite -6 and to govern product specificity for G6 production.

Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS-PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed. These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins. We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome. The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four. Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as 35S-methionine or the stable isotope, 15N.

Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site.

Cyclodextrin glycosyltransferase (CGTase) belonging to the alpha-amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related alpha-1,4-glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in alpha-amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose-complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y). The temperature factors of the region 259-269 in native F283L increased >10 A(2) compared with the wild type. The complex formation with acarbose not only increased the temperature factors (>10 A(2)) but also changed the structure of the region 257-267. This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding. The conformation of the side-chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259. The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range. The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pK(a) of Glu257 in F283Y may be lower than that in the wild type.

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011.

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.

Identification and characterization of the Bacillus subtilis D-glucarate/galactarate utilization operon ycbCDEFGHJ.

In the course of the Bacillus subtilis functional genomics project, an open reading frame called ycbG whose product is classified as a transcriptional regulatory protein with a helix-turn-helix motif in the putative D-glucarate/galactarate utilization operon (ycbCDEFGHJ) was initially screened as the gene disruptant that exhibits a defect that blocked the early stage of sporulation. However, the transcription of ycbCDEFG was extremely highly induced in response to nutrient exhaustion by the disruption of ycbG, but inactivation of the transcription from upstream ycbC in the ycbG mutant restored the sporulation efficiency, suggesting that the inappropriate over-production of the ycbCDEFG gene products inhibits efficient sporulation. We further analyzed the role of the ycbCDEFGHJ cluster and found that (i) a unit of ycbCDEFGHJ was induced by either D-glucarate or D-galactarate, and (ii) the cell growth was inhibited by the mutation of the ycbF and ycbH genes, that respectively encode the putative proteins, D-glucarate dehydratase and D-galactarate dehydratase on plates supplemented with D-glucarate and D-galactarate, respectively, as the sole carbon source. Our results indicate that the ycbCDEFGHJ genes are involved in the utilization of D-glucarate and D-galactarate in B. subtilis.

Expression of a small RNA, BS203 RNA, from the yocI-yocJ intergenic region of Bacillus subtilis genome.

We isolated and characterized a novel small RNA from Bacillus subtilis. We termed this molecule BS203 RNA from the length of its mature form (203 nt) and located the corresponding gene at the yocI-yocJ intergenic region on the B. subtilis genome. Northern blotting revealed that it is transcribed in vegetative growing cells and that the amount of BS203 RNA decreased in the middle of the vegetative phase. A computer-aided prediction of the BS203 RNA secondary structure revealed three characteristic stem-loop structures. Despite active expression during the vegetative phase, growth of the knockout mutant was not affected by depletion of BS203 RNA. A phylogenetic comparison of the sequence of the BS203 RNA with other Bacillus species including B. cereus and B. halodurans C-125, or Clostridium perfringens suggests that the sequence is unique to Bacillus subtilis.

Microbial diversity with dominance of 16S rRNA gene sequences with high GC contents at 74 and 98 °C subsurface crude oil deposits in Japan.

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.

Quantitation of de novo localized (15)N-labeled lipoproteins and membrane proteins having one and two transmembrane segments in a Bacillus subtilis secA temperature-sensitive mutant using 2D-PAGE and MALDI-TOF MS.

We developed a means of quantifying proteins that have just localized in the cytoplasmic membrane using 15N-whole cell labeling together with 2D-PAGE and MALDI-TOF MS. The localization of 18 among 20 proteins consisting of 8 lipoproteins, 11 integral membrane proteins having one or two transmembrane segments and one secretory protein in the membrane fractions of Bacillus subtilis, was inhibited by the absence of SecA in a temperature-sensitive mutant. The time course of inhibition indicated that SecA participates in the localization of those proteins through immediately dependent, delayed dependent, and independent ways.

Protein traffic for secretion and related machinery of Bacillus subtilis.

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.

A 32 kb nucleotide sequence from the region of the lincomycin-resistance gene (22 degrees-25 degrees) of the Bacillus subtilis chromosome and identification of the site of the lin-2 mutation.

A 32 kb nucleotide sequence in the region of the lincomycin-resistance gene, located from 22 degrees to 25 degrees on the Bacillus subtilis chromosome, was determined. Among 32 putative ORFs identified, four [lipA for lipase, natA, natB and yzaE (renamed yccK)] have already been reported, although the functions of NatA, NatB and YccK remain to be characterized. Six putative products were found to exhibit significant similarity to known proteins in the databases, namely L-asparaginase precursor, protein aspartate phosphatase, alpha-glucosidase, two tellurite-resistance proteins and a hypothetical protein from B. subtilis. The region of the tellurite-resistance gene, consisting of seven ORFs, seems to correspond to an operon. The products of 14 ORFs exhibited considerable or limited similarity to known proteins. The sequenced region seems to be rich in membrane proteins, since at least 16 gene products appeared to contain membrane-spanning domains. The site of the lin-2 mutation (two nucleotide replacements) was mapped and identified by sequencing. This site is located between a putative promoter and the SD sequence of ImrA (yccB) [a putative repressor of the lmr operon, which consists of lmrA and lmrB (yccA)]. LmrB is a homologue of proteins involved in drug-export systems and seems likely to be the protein responsible for resistance to lincomycin.

Biochemical and crystallographic analyses of maltohexaose-producing amylase from alkalophilic Bacillus sp. 707.

Maltohexaose-producing amylase, called G6-amylase (EC 3.2.1.98), from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) from starch and related alpha-1,4-glucans. To elucidate the reaction mechanism of G6-amylase, the enzyme activities were evaluated and crystal structures were determined for the native enzyme and its complex with pseudo-maltononaose at 2.1 and 1.9 A resolutions, respectively. The optimal condition for starch-degrading reaction activity was found at 45 degrees C and pH 8.8, and the enzyme produced G6 in a yield of more than 30% of the total products from short-chain amylose (DP = 17). The crystal structures revealed that Asp236 is a nucleophilic catalyst and Glu266 is a proton donor/acceptor. Pseudo-maltononaose occupies subsites -6 to +3 and induces the conformational change of Glu266 and Asp333 to form a salt linkage with the N-glycosidic amino group and a hydrogen bond with secondary hydroxyl groups of the cyclitol residue bound to subsite -1, respectively. The indole moiety of Trp140 is stacked on the cyclitol and 4-amino-6-deoxyglucose residues located at subsites -6 and -5 within a 4 A distance. Such a face-to-face short contact may regulate the disposition of the glucosyl residue at subsite -6 and would govern the product specificity for G6 production.

Lincomycin resistance mutations in two regions immediately downstream of the -10 region of lmr promoter cause overexpression of a putative multidrug efflux pump in Bacillus subtilis mutants.

We isolated 19 lincomycin-resistant Bacillus subtilis mutants by expressing lmrB encoding a putative multidrug efflux protein. Eighteen of the mutants altered at two regions (-3 to -1 and +15) immediately downstream of the -10 region of the lmr promoter increased lmr transcription in vivo and in vitro.

Expression of the ftsY gene, encoding a homologue of the alpha subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis.

Bacillus subtilis FtsY (Srb) is a homologue of the alpha subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor sigma(A). During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of sigma(K) and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a sigma(K)- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.

Multidrug resistant phenotype of Bacillus subtilis spontaneous mutants isolated in the presence of puromycin and lincomycin.

Spontaneous mutants were isolated by growing Bacillus subtilis 168 in the presence of high concentrations of puromycin and lincomycin. These mutants showed increased resistance to several drugs other than these two drugs. The ImrAB genes, which encode a transcriptional repressor and a drug efflux protein of the major facilitator superfamily, were involved in this phenotype. Northern hybridization analysis showed that the expression of ImrAB gene increased more than 30-fold. The following two types of mutations were found to be responsible for the multidrug resistant phenotype: (i) a nucleotide replacement in the region between the promoter and initiation codon of ImrA and (ii) nucleotide replacements that resulted in amino acid replacements in the LmrA protein. The results indicate that LmrB is a multidrug resistant protein and that LmrA is a repressor, which autogenously represses the transcription of the ImrAB operon.