Bacillus Halodurans C-125 Research Articles

Combinatorial optimization of the hybrid cellulase complex structure designed from modular libraries.

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes. Therefore, we designed hybrid cellulase complexes from 49 biotinylated catalytic domain (CD) and 30 biotinylated cellulose-binding domain (CBD) libraries on streptavidin-conjugated nanoparticles to enhance cellulose hydrolysis by mimicking the cellulosome structure. The hybrid cellulase complex, incorporating both native CD and CBD, significantly improved reducing sugar production from cellulose compared to free native modular enzymes. The optimal CBD for each hybrid cellulase complex differed from that of the native enzyme. The most effective hybrid cellulase complex was observed with the combination of CD6-4 from Thermobifida fusca YX and CBD46 from the Bacillus halodurans C-125. The hybrid cellulase complex/CD6-4-CBD46 and -CD6-4-CBD2-5 combinations showed increased reducing sugar production. Similar results were also observed in microcrystalline cellulose degradation. Furthermore, clustering on nanoparticles enhanced enzyme thermostability. Our results demonstrate that hybrid cellulase complex structures improve enzyme function through synergistic effects and extend the lifespan of the enzyme.

Characteristics of the ErmK Protein of Bacillus halodurans C-125.

Bacillus halodurans C-125 is an alkaliphilic microorganism that grows best at pH 10 to 10.5. B. halodurans C-125 harbors the erm (erythromycin resistance methylase) gene as well as the mphB (macrolide phosphotransferase) and putative mef (macrolide efflux) genes, which confer resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. The Erm protein expressed in B. halodurans C-125 could be classified as ErmK because it shares 66.2% and 61.2% amino acid sequence identity with the closest ErmD and Erm(34), respectively. ErmK can be regarded as a dimethylase, as evidenced by reverse transcriptase analysis and the antibiotic resistance profile exhibited by E. coli expressing ermK. Although ErmK showed one-third or less in vitro methylating activity compared to ErmC', E. coli cells expressing ErmK exhibited comparable resistance to erythromycin and tylosin, and a similar dimethylation proportion of 23S rRNA due to the higher expression rate in a T7 promoter-mediated expression system. The less efficient methylation activity of ErmK might reflect an adaption to mitigate the fitness cost caused by dimethylation through the Erm protein presumably because B. halodurans C-125 has less probability to encounter the antibiotics in its favorable growth conditions and grows retardedly in neutral environments. IMPORTANCE Erm proteins confer MLSB antibiotic resistance (minimal inhibitory concentration [MIC] value up to 4,096 μg/mL) on microorganisms ranging from antibiotic producers to pathogens, imposing one of the most pressing threats to clinics. Therefore, Erm proteins have long been speculated to be plausible targets for developing inhibitor(s). In our laboratory, it has been noticed that there are variations in enzymatic activity among the Erm proteins, Erm in antibiotic producers being better than that in pathogens. In this study, it has been observed that Erm protein in B. halodurans C-125 extremophile is a novel member of Erm protein and acts more laggardly, compared to that in pathogen. While this sluggishness of Erm protein in extremophile might be evolved to reduce the fitness cost incurred by Erm activity adapting to its environments, this feature could be exploited to develop the more potent and/or efficacious drug to combat formidably problematic antibiotic-resistant pathogens.

Isolation of An Erythromycin-Resistant (Putative) Gene From Bacillus Halodurans CM1

Bacillus halodurans CM1 is one of the bacterial strains from BPPT Culture Collection which had been isolated from sediments in the Cimanggu hot spring, West Java, previously. The whole genomic DNA of this strain has not yet been deeply studied despite its potential as host to produce several kinds of useful enzymes, such as xylanase, lipase, and protease. Besides the industrial enzymes, potential genes in B. halodurans CM1, for example antibiotic-resistant genes, could be engineered later for further purposes. Earlier experiments showed that CM1 strain has the ability to survive in the media with erythromycin, suggesting there might be genes responsible for this erythromycin resistance. This study aimed to isolate erythromycin-resistant gene from B. halodurans CM1 using a PCR approach. The primers used for PCR were designed based on the GenBank database of whole genome Bacillus halodurans C-125. The PCR product was then ligated into a pGEM-T easy cloning vector, and the recombinant vector then transformed into Escherichia coli DH5a. The DNA fragment with the size of 864 bp was obtained and it encoded 288 deduced amino acids. DNA sequence analysis showed that the gene had 99% similarity with gene encoding erythromycin resistance from B. halodurans C-125 (GenBank No access: BA000004.3, ErmK). There are 3 nucleotides in the ErmK sequence of B. halodurans CM1 that are different from that of another well-known strain B. halodurans C-125. This dissimilar pattern of sequence encodes Leu instead of Ser in 155th orders of CM1 amino acid sequence.

Assessment of a Potential Role of Dickeya dadantii DSM 18020 as a Pectinase Producer for Utilization in Poultry Diets Based on in silico Analyses.

Currently, the poultry industry has been faced with consumer pressure to utilize only vegetable feedstuffs in poultry diets, eliminate antibiotics from poultry production, and rear poultry in free range systems. To maintain current production standards, the industry must determine ways to enhance nutrient uptake and utilization further. One possible solution is the supplementation of pectinase, an enzyme that degrades pectin within the cell walls of plants, in poultry diets. Therefore, the objective of the current study was to determine the potential role of a pectinase producer, Dickeya dadantii DSM 18020, as a commercially utilized pectinase producer in poultry diets against other known pectinase producers, in silico. In the current study, whole genomes of Dickeya dadantii DSM 18020 (Dd18020), D. dadantii 3937 (Dd3937), D. solani IPO 2222 (Ds2222), Bacillus halodurans C-125 (BhC125), and B. subtilis subsp. subtilis str. 168 (Bs168) were compared using bioinformatic approaches to compare the chromosomal genome size, GC content, protein coding genes (CDS), total genes, average protein length (a.a.) and determine the predicted metabolic pathways, predicted pectin degrading enzymes, and pectin-degradation pathways across pectinase producers. Due to insufficient information surrounding the genome of Dd18020 (lack of annotation), the genome of Dd3937, a 99% identical genome to Dd18020, was utilized to compare pectinase-associated enzymes and pathways. The results from the current study demonstrated that Dd3937 possessed the most significant proportion of pathways presented and the highest number of pathways related to degradation, assimilation, and utilization of pectin. Also, Dd18020 exhibited a high number of pectinase-related enzymes. Both Dd3937 and Dd2222 shared the pectin degradation I pathway via the EC 3.1.1.11, EC 3.2.1.82, and EC 4.2.2.- enzymes, but did not share this pathway with either Bacillus species. In conclusion, Dd18020 demonstrated the genetic potential to produce multiple pectinase enzymes that could be beneficial to the degradation of pectin in poultry diets. However, for Dd18020 to become a commercially viable enzyme producer for the poultry industry, further research quantifying the pectinase production in vitro and determining the stability of the produced pectinases during feed manufacturing are necessary.

Evaluation of the ability of alkalophilic bacteria to form a biofilm on the surface of Portland cement-based mortars

This paper investigates bacteria colonisation through biofilm formation, based on the premise that biofilm helps bacteria to have a better development. The aim is to homogenize bacterial growth on recycled concrete aggregates (RCA) to obtain a homogeneous precipitation of calcium carbonate (CaCO3). In previous studies, Bacillus halodurans C-125 was selected to perform biocarbonation on RCA to generate a coat of CaCO3 and diminish water absorption. Contrary to expectations, its poor development led to an heterogeneous CaCO3 precipitation, resulting in an inefficient treatment. Within the framework of this criterion the genetic information of B. halodurans C-125 was compared with a homologous specie “Bacillus subtilis str. 168” to know if it possessed the genes to encode Tas A and Tap A proteins. These proteins consolidate a robust biofilm in Bacillus subtilis str. 168, which promotes bacterial development and adhesion to a surface. Remarkably, B. halodurans C-125 lacks the genes to produce Tas A and Tap A. B. halodurans C-125 was also compared with a group of bacteria isolated from RCA to produce biofilm on MSgg media. Curiously, B. halodurans C-125 did not form a robust biofilm while the bacteria isolated from RCA did. Because of the capacity of the isolated bacteria to form biofilm, they were inoculated on a mortar disk with nutrient and MSgg broth. The results showed traces of bacterial development and precipitation of CaCO3 in form of calcite.

Functional characterization of hormone sensitive-like lipase from Bacillus halodurans: synthesis and recovery of pNP-laurate with high yields.

The HSL-like lipase encoding gene (Blip) from the polyextremophile Bacillus halodurans C-125 has been heterologously expressed in E.coli BL21(DE3). The enzyme is a monomer of ~42kDa. It has extremely high thermal stability with a t 1/2 of 35min at 100°C. Thermal denaturation/renaturation studies by CD and fluorescence analysis revealed complete refolding of the protein back to its native conformation even after 30min at 90°C. Blip prefers substrates with mid to long chain fatty acids. It has a higher catalytic efficiency on para-nitrophenyl fatty acyl esters as opposed to triacylglycerides (k cat/K m with pNP-palmitate as a substrate was 2.52×105 mM-1min-1 while that with glyceryl tripalmitin was 4.06×102 mM-1min-1, respectively). The enzyme also has a unique selectivity for hydrolysis of unsaturated fatty acyl esters. The enzyme catalyses the synthesis of pNP-laurate with an optimized conversion of 95.94±0.24%. A simple procedure for purification of the product has been developed that led to 89.91±0.33% product recovery.

Redesigning pH optimum of Geobacillus sp. TF16 endoxylanase through in silico designed DNA swapping strategy

Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications.The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes.

4-O-Methyl Modifications of Glucuronic Acids in Xylans Are Indispensable for Substrate Discrimination by GH67 α-Glucuronidase from Bacillus halodurans C-125.

A GH67 α-glucuronidase gene derived from Bacillus halodurans C-125 was expressed in E. coli to obtain a recombinant enzyme (BhGlcA67). Using the purified enzyme, the enzymatic properties and substrate specificities of the enzyme were investigated. BhGlcA67 showed maximum activity at pH 5.4 and 45 °C. When BhGlcA67 was incubated with birchwood, oat spelts, and cotton seed xylan, the enzyme did not release any glucuronic acid or 4-O-methyl-glucuronic acid from these substrates. BhGlcA67 acted only on 4-O-methyl-α-D-glucuronopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (MeGlcA3Xyl3), which has a glucuronic acid side chain with a 4-O-methyl group located at its non-reducing end, but did not on β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronopyranosyl-(l→2)]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylop- yranose (MeGlcA3Xyl4) and α-D-glucuronopyranosyl-(l→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (GlcA3Xyl3). The environment for recognizing the 4-O-methyl group of glucuronic acid was observed in all the crystal structures of reported GH67 glucuronidases, and the amino acids for discriminating the 4-O-methyl group of glucuronic acid were widely conserved in the primary sequences of the GH67 family, suggesting that the 4-O-methyl group is critical for the activities of the GH67 family.

Updating microbial genomic sequences: improving accuracy & innovation

BackgroundMany bacterial genome sequences completed using the Sanger method may contain assembly errors due in-part to low sequence coverage driven by cost.FindingsTo illustrate the need for re-sequencing of pre-nextgen genomes and to validate sequenced genomes, we conducted a series of experiments, using high coverage sequencing data generated by a Illumina Miseq sequencer to sequence genomic DNAs of Bacteroides fragilis NCTC 9343, Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150, Vibrio cholerae O1 biovar El Tor str. N16961, Bacillus halodurans C-125 and Caulobacter crescentus CB15, which had previously been sequenced by the Sanger method during the early 2000’s.ConclusionsThis study revealed a number of discrepancies between the published assemblies and sequence read alignments for all five bacterial species, suggesting that the continued use of these error-containing genomes and their genetic information may contribute to false conclusions and/or incorrect future discoveries when they are used.

Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation

BackgroundDNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.ResultsWe characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.ConclusionsWe have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.

The relationship between a coiled morphology and Mbl in alkaliphilic Bacillus halodurans C-125 at neutral pH values

The facultative alkaliphilic Bacillus halodurans C-125 can grow in a pH range from 6.8 to 10.8. The morphology of the cells grown at pH values above 7.5 is rod shaped, whereas, that gown at pH values less than 7.5 is coiled. Cytoplasmic membrane staining revealed that this coiled morphology was formed not by one filamentous cell, but by many chained bent/non-bent cells. Prokaryotic actin and tubulin homologs (MreB, Mbl MreBH, and FtsZ, respectively) are known to function as bacterial cytoskeleton proteins. The transcription levels of ftsZ, mreB, and mreBH genes were hardly affected by growth pH. However, the level of the mbl gene was significantly decreased at neutral pH values. Moreover, the expression level of the Mbl protein at pH 7.0 was about one-fourth of that at pH 10. Immunofluorescence microscopy (IFM) showed that the Mbl protein was localized as a helical structure in the rod-shaped cell grown at pH 10, whereas a helical structure was not observed in the cells grown at pH 7.0. Fluorescent vancomycin staining showed insertion of new peptidoglycan strands of sidewalls occurred in the cells grown at pH 7.0. These data suggested that a decrease in the expression level of the Mbl protein can influence the morphology of B. halodurans C-125 grown at pH 7.0 without influencing insertion of new peptidoglycan strands.

Characterization of a New Thermoalkalophilic Xylanase-Producing Bacterial Strain Isolated from Cimanggu Hot Spring,West Java, Indonesia

Alkalophilic bacteria and their enzymes are important for industrial applications. Therefore, finding out new strains of alkalophilic bacteria from Indonesian microbial diversity is still required. In this study, a thermoalkalophilic bacterium was isolated from sediment of local hot spring, Cimanggu,West Java. The temperature of the spot was 60 °C and the pH was 8. The bacterium could live at pH 11 and temperature 55 °C, and produced xylanase that have optimal activity at alkaline pH 9 and high temperature 70 °C. Based on the analyses of 16S rRNA sequence similarity and biochemical characteristics, this strain was clustered into the same group of Bacillus halodurans species with 99% identity to Bacillus halodurans C-125. The isolate also showed other enzyme activities such as amylase, protease, and gelatinase, promising its potential use as an industrial enzymes producer.

Improved genetic transformation methods for the model alkaliphile Bacillus halodurans C-125

Bacillus halodurans C-125 is a Gram-positive bacterium that was the first alkaliphilic species to have its genome completely sequenced. Despite its many years as a model for alkaliphily and source of industrially important enzymes, genetic manipulation of B. halodurans C-125 remains difficult, and therefore, we sought to develop a robust method to allow routine transformation of this organism. A plasmid artificial modification system (PAM system, Yasui et al. 2008) for B. halodurans C-125 was created that increases transformation efficiency by 10- to 1000-fold. Also, recovering transformed protoplasts on succinate nutrient agar (SNA) yields faster, more robust colony recovery than on the traditional recovery medium. Combining these two techniques often allows recovery of transformants in as little as 48 h. Use of the B. halodurans C-125 PAM system and SNA greatly improves the efficiency and speed of protoplast transformation of B. halodurans C-125. These techniques allow routine genetic manipulation of B. halodurans C-125, a model alkaliphilic bacterium with important industrial properties.

Antibacterial activity of cyclodextrins against Bacillus strains

Growth of alkaliphilic Bacillus halodurans C-125 both on agar plates and in liquid culture was inhibited by methyl-beta-cyclodextrin (CD). Furthermore, resting cells of the strain were lysed by contact with methyl-beta-CD higher than 10 mM. alpha-CD also showed lysis activity against Bacillus and related strains. The activity was not observed with Gram-negative and Gram-positive bacteria except for Bacillus strains. Fluorescence staining and scanning electron microscopy of cells revealed that methyl-beta-CD disrupted cell membranes, and consequently, the cells were lysed. This is a novel physiological property of CDs.

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na +/H + antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na +-resistance on an antiporter-deficient E. coli strain, nor did they confer Na +/H + antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.

Molecular Anatomy of the Alkaliphilic Xylanase from Bacillus halodurans C-125

Two regions in xylanase A from Bacillus halodurans C-125 (XynA), an alkaliphilic xylanase, were identified to be responsible for its activity at basic pH by comparing the dissociation constants of the XynA proton donor Glu residue (pK(e2) and pK(es2)) with those of xylanase B from Clostridium stercorarium F9 (XynB) and their mutants constructed by substituting either Ser137/Asn127 of XynA/XynB or the 4th loop, designed based on the structural difference close to the proton donor. The substitution of XynB at Asn127 into Ser increased pK(e2) by 0.37. The effect is explained that the positive charge of His126 likely affects the proton donor via Asn127 and a water molecule in XynB, resulting in a decrease in pK(e2), whereas such interactions were not observed with Ser. The substitution of XynB at the 4th loop into XynA (XynB Loop4A) increased the pK(e2) and pK(es2) values by 0.29 and 0.62, respectively. The effect of the 4th loop in XynA is likely due to a hydrogen bond between Asp199 in the loop and Tyr239, which interacts with both the proton donors Glu195 and Arg204, with flexibility of the loop. Both the mutations independently affected the increases in pK(e2).

Cloning and characterization of dihydrofolate reductase from a facultative alkaliphilic and halotolerant bacillus strain

Elucidation of the molecular basis of the stability of enzymes from extremophilic organisms is of fundamental importance for various industrial applications. Due to the wealth of structural data from various species, dihydrofolate reductase (DHFR, EC 1.5.1.3) provides an excellent model for systematic investigations. In this report, DHFR from alkaliphilic Bacillus halodurans C-125 was cloned and expressed in E. coli. Functional analyses revealed that BhDHFR exhibits the most alkali-stable phenotype of DHFRs characterized so far. Optimal enzyme activity was observed in a slightly basic pH region ranging from 7.25 to 8.75. Alkali-stability is associated with a remarkable resistance to elevated temperatures (half-life of 60 min at 52.5 degrees C) and to high concentrations of urea (up to 3 M). Although the secondary structure shows distinct similarities to those of mesophilic DHFR molecules, BhDHFR exhibits molecular features contributing to its alkaliphilic properties. Interestingly, the unique phenotype is diminished by C-terminal addition of a His-tag sequence. Therefore, His-tag-derivatized BhDHFR offers the opportunity to obtain deeper insights into the specific mechanisms of alkaliphilic adaption by comparison of the three dimensional structure of both BhDHFR molecules.

A Family 8 Glycoside Hydrolase from Bacillus halodurans C-125 (BH2105) Is a Reducing End Xylose-releasing Exo-oligoxylanase

The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exo-splitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the beta-anomer of the substrate to form beta-xylose and alpha-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2-7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two alpha-arabinofuranosidases, one alpha-glucuronidase, and three beta-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.

Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less β-lactamase (′ bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The ′ bla gene was cloned into a bacteriophage Mu minitransposon enabling translational fusions between ′ bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library to be tagged. It is suited for identifying genes encoding extracytosolic proteins in gene libraries of a wide range of prokaryotic organisms.

Characterization of endo-beta-N-acetylglucosaminidase from alkaliphilic Bacillus halodurans C-125.

The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-beta-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids. Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-beta-N-acetylglucosaminidase activity. Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity. On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced. These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity. Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-beta-N-acetylglucosaminidases of various origins.