A Novel Gene Required for the Alkaliphily of the Facultative Alkaliphile Bacillus sp. Strain C-125

This study clarifies the effect of exposure to cigarette smoke on L-ascorbic acid (AsA) metabolism and on the activities of drug-metabolizing enzymes. Male Wistar rats were used. The test rats (group T) were exposed to sidestream smoke from cigarette for 2 h every day for 25 days. During the experimental period, the excreted amount of AsA in the urine from group T was higher than that from the control group (group C). At the end of the experimental period, the AsA content of the plasma and tissues, the liver cytochrome P-450 content and the activities of drug-metabolizing enzymes in group T were each higher than those in group C.     

Decrease in cytoplasmic pH-homeostastatic activity of the alkaliphile Bacillus lentus C-125 by a cell wall defect

Cytoplasmic pH homeostatic activities of cell wall-defective derivatives of the alkaliphile Bacillus lentus C-125 were assessed using a pH-sensitive fluorescent probe, BCECF. It was shown that the acidic cell wall components took part in maintenance of the cytoplasmic pH neutrality at alkaline pH.     

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.     

Relative Contribution of the Cell Wall, Cytoplasmic Membrane, and Cytoplasm to the Gram-Positive Characteristic of Bacillus megaterium.

Bartholomew, J. W. (University of Southern California, Los Angeles), and Thomas Cromwell. Relative contribution of the cell wall, cytoplasmic membrane, and cytoplasm to the gram-positive characteristic of Bacillus megaterium. J. Bacteriol. 90:643-647. 1965.-A comparison of the roles of the cell wall, cytoplasmic membrane, and cytoplasmic components revealed that the intact cell wall was the dominant contributor to the gram-positive state. Protoplasts of Bacillus megaterium were confirmed as being gram-negative, as reported by Gerhardt et al. The "gram-positive protoplast" report of Amano et al. was shown to be a laboratory-produced artifact, resulting from the comparison of smears made from saline suspensions of Escherichia coli cells with smears made from formalin-sucrose suspensions of B. megaterium protoplasts.     

Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125

Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank. The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes. One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported. The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B. halodurans C-125 and B. subtilis. Received: March 26, 1999 / Accepted: April 27, 1999     

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).     

Cell Wall Protein in Bacillus subtilis

The cell wall of Bacillus subtilis 168 contains protein that is refractory to removal by salts, detergents, and denaturants.     

Alterations in Auxin Homeostasis Suppress Defects in Cell Wall Function

The plant cell wall is a highly dynamic structure that changes in response to both environmental and developmental cues. It plays important roles throughout plant growth and development in determining the orientation and extent of cell expansion, providing structural support and acting as a barrier to pathogens. Despite the importance of the cell wall, the signaling pathways regulating its function are not well understood. Two partially redundant leucine-rich-repeat receptor-like kinases (LRR-RLKs), FEI1 and FEI2, regulate cell wall function in Arabidopsis thaliana roots; disruption of the FEIs results in short, swollen roots as a result of decreased cellulose synthesis. We screened for suppressors of this swollen root phenotype and identified two mutations in the putative mitochondrial pyruvate dehydrogenase E1α homolog, IAA-Alanine Resistant 4 (IAR4). Mutations in IAR4 were shown previously to disrupt auxin homeostasis and lead to reduced auxin function. We show that mutations in IAR4 suppress a subset of the fei1 fei2 phenotypes. Consistent with the hypothesis that the suppression of fei1 fei2 by iar4 is the result of reduced auxin function, disruption of the WEI8 and TAR2 genes, which decreases auxin biosynthesis, also suppresses fei1 fei2. In addition, iar4 suppresses the root swelling and accumulation of ectopic lignin phenotypes of other cell wall mutants, including procuste and cobra. Further, iar4 mutants display decreased sensitivity to the cellulose biosynthesis inhibitor isoxaben. These results establish a role for IAR4 in the regulation of cell wall function and provide evidence of crosstalk between the cell wall and auxin during cell expansion in the root.     

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

Aims: 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. Methods and Results: 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. Conclusions: 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. Significance and Impact of the Study: These techniques allow routine genetic manipulation of B. halodurans C‐125, a model alkaliphilic bacterium with important industrial properties.     

Presence of fucosamine in teichuronic acid of the alkalophilic Bacillus strain C-125.

Cell walls of the alkalophilic Bacillus strain C-125 are composed of gamma-peptidoglycan, teichuronic acid and a polymer of glucuronate and glutamate. An amino sugar that was a main component of the teichuronic acid did not correspond to any of the commercially available hexosamines. The amino sugar was purified into crystalline form from the hydrolysate of the teichuronic acid by ion-exchange chromatography and then partition chromatography on a cellulose column. The amino sugar was identified as D-fucosamine (2-amino-2,6-dideoxy-D-galactose) by 400 MHz n.m.r. spectrometric analysis, measurement of optical rotation and elemental analysis.     

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.     

Cell Wall and Morphological Changes Induced by Temperature Shift in Bacillus subtilis Cell Wall Mutants

Bacillus subtilis RUB1012 and RUB1013 have the following phenotype when grown at 45 degrees C: no growth on tryptose blood agar base, growth as clumps of spheres in broth culture, a slow autolysis rate, and a low proportion of teichoic acid to peptidoglycan. Revertants of strain RUB1012 (RUB2032, RUB2012, and RUB2042) that could grow on tryptose blood agar base were isolated. Each revertant had a different proportion of teichoic acid to peptidoglycan. The nanomoles of phosphorus per milligram of cell wall at the nonpermissive temperature were 141, 160, 236, and 541 for strain RUB1012 and revertants RUB2032, 2012, and 2042, respectively, as compared with 1,100 for the parent strain. With most bacteriophage tested, plating efficiency was related to the amount of glucosylated teichoic acid. Scanning electron microscopy was used to study strain RUB2032 during a shift from growth at 30 degrees C to growth at 45 degrees C. The change from rod to sphere began with the thickening of the cylindrical portion of the cell. Caps of the cells appeared to be immune to the thickening process. During growth, the cells became progressively shorter and thicker, and cell separation was inhibited. When cells of strain RUB2032 were shifted from growth at 45 degrees C to growth at 30 degrees C, accumulation of an amorphous material on the outer surfaces of the cells preceded the change from sphere to rod morphology. Cells remained clumped, with rods appearing at the periphery of the clumps. Analysis by DNA-mediated transformation and PBS1-mediated transduction indicated that strains RUB1012 and RUB1013 have multiple mutations mapping in the same region as other cell wall mutations.     

Molecular Sieving by the Bacillus megaterium Cell Wall and Protoplast

Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size. Weight per cent uptake values (R(w)) were measured at diffusional equilibrium under conditions that negated the influences of adsorption or active transport. Plots of R(w) for intact cells as a function of number-average molecular weight ( M(n)) or Einstein-Stokes hydrodynamic radius ( r(ES)) of the solutes showed three phases: a protoplast uptake phase with a polydisperse exclusion threshold of M(n) = 0.6 x 10(3) to 1.1 x 10(3), r(ES) = 0.6 to 1.1 nm; a cell wall uptake phase with a polydisperse exclusion threshold of M(n) = 0.7 x 10(5) to 1.2 x 10(5), r(ES) congruent with 8.3 nm; and a total exclusion phase. Isolated cell walls showed only the latter two phases. However, it became evident that the cell wall selectively passed only the smallest molecules in a heterodisperse polymer sample. When the molecular-weight distributions of polyglycol samples ( M(n) = 1,000, 1,450, and 3,350) were determined by analytical gel chromatography before and after uptake by intact cells or isolated cell walls, a quasi-monodisperse exclusion threshold was obtained corresponding to M(n) = 1,200, r(ES) = 1.1 nm. The permeability of isolated protoplasts was assessed by the relative ability of solutes to effect osmotic stabilization. An indefinite exclusion threshold, evident even with monodisperse sugars, was attributed to lengthwise orientation of the penetrating rod-shaped molecules. Altogether, the best estimate of the limiting equivalent porosity of the protoplast was 0.4 to 0.6 nm in radius and of the cell wall, 1.1 nm.     

Identification and distribution of new insertion sequences in the genome of alkaliphilic Bacillus halodurans C-125.

Fifteen kinds of new insertion sequences (ISs), IS641 to IS643, IS650 to IS658, IS660, IS662, and IS663, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic Bacillus halodurans C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS650, IS653, IS660, and IS663 generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS650, IS653, and IS657 carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS642, IS643, IS654, IS655, IS657, and IS658) belong to a separate IS family (IS630, IS21, IS256, IS3, IS200/IS605, and IS30, respectively) as a new member. Also, IS651 and IS652 were characterized as new members of the ISL3 family. Significant similarity was found between the transposase (Tpase) sequences between IS650 and IS653 (78.2%), IS651 and IS652 (56.3%), IS656 and IS662 (71.0%), and IS660 and IS663 (44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS651 in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and L-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.     

嗜碱芽孢杆菌C-125木糖苷酶基因的表达与酶特征鉴定

嗜碱芽孢杆菌(Bacillus halodurans)C-125菌株的基因组中,一个编码木糖苷酶的基因(BH1068)被克隆并在大肠杆菌中获得高效表达。通过全面分析纯化蛋白,确证了它的木糖苷酶功能。该酶在pH4～9的范围内保持稳定,最适pH值为中性,有较宽的最适温度(35°C～45°C),且能在45°C范围内保持稳定。这些特性使得该酶可在较为宽广的条件下对木聚糖进行酶促降解。该酶对人工合成底物对硝基苯-β-木糖苷(p-nitrophenyl-β-xylose,pNPX)的比活力为174mU/mg蛋白质,且木糖对其反馈抑制较弱(抑制常数Ki为300mmol/L)。结果显示该酶是活性较高且较耐木糖抑制的细菌源木糖苷酶。该酶与商品化的木聚糖酶一起水解山毛举木聚糖(Beechwood xylan)时显示了增效作用,且水解率可获40%。该酶最适pH为中性,对木糖耐受等特性与大多数来源于真菌、最适pH为酸性、对木糖敏感的木糖苷酶将有较好的互补。结果表明该酶在木聚糖或含木聚糖多糖的单糖化过程可能发挥重要作用。     

Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25 Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region. Received: May 6, 1998 / Accepted: May 26, 1998     

The BH1999 protein of Bacillus halodurans C-125 is gentisyl-coenzyme A thioesterase

In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a k(cat)/K(m) of 1.6 x 10(6) M(-1) s(-1) and the hydrolysis of 3-hydroxybenzoyl-CoA with a k(cat)/K(m) of 3.0 x 10(5) M(-1) s(-1). All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.