Growth and productivity of rice are negatively affected by soil salinity. However, some salt-tolerant rhizosphere-inhabiting bacteria can improve salt resistance of plants, thereby augmenting plant growth and production. Here, we isolated a total of 53 plant-growth-promoting rhizobacteria (PGPR) from saline and non-saline areas in Bangladesh where electrical conductivity was measured as >7.45 and <1.80 dS/m, respectively. Bacteria isolated from saline areas were able to grow in a salt concentration of up to 2.60 mol/L, contrary to the isolates collected from non-saline areas that did not survive beyond 854 mmol/L. Among the salt-tolerant isolates, Bacillus aryabhattai, Achromobacter denitrificans, and Ochrobactrum intermedium, identified by comparing respective sequences of 16S rRNA using the NCBI GenBank, exhibited a higher amount of atmospheric nitrogen fixation, phosphate solubilization, and indoleacetic acid production at 200 mmol/L salt stress. Salt-tolerant isolates exhibited greater resistance to heavy metals and antibiotics, which could be due to the production of an exopolysaccharide layer outside the cell surface. Oryza sativa L. fertilized with B. aryabhattai MS3 and grown under 200 mmol/L salt stress was found to be favoured by enhanced expression of a set of at least four salt-responsive plant genes: BZ8, SOS1, GIG, and NHX1. Fertilization of rice with osmoprotectant-producing PGPR, therefore, could be a climate-change-preparedness strategy for coastal agriculture.
A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.
Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium-proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.
This study investigated the capability of a biosurfactant produced by a novel strain of Bacillus salmalaya to enhance the biodegradation rates and bioavailability of organic contaminants. The biosurfactant produced by cultured strain 139SI showed high physicochemical properties and surface activity in the selected medium. The biosurfactant exhibited a high emulsification index and a positive result in the drop collapse test, with the results demonstrating the wetting activity of the biosurfactant and its potential to produce surface-active molecules. Strain 139SI can significantly reduce the surface tension (ST) from 70.5 to 27 mN/m, with a critical micelle concentration of 0.4%. Moreover, lubricating oil at 2% (v/v) was degraded on Day 20 (71.5). Furthermore, the biosurfactant demonstrated high stability at different ranges of salinity, pH, and temperature. Overall, the results indicated the potential use of B. salmalaya 139SI in environmental remediation processes.
Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
The effectiveness of β-lactam antibiotics as chemotherapeutic agents to treat bacterial infections is gradually threatened with the emergence of antibiotic resistance mechanism among pathogenic bacteria through the production metallo-β-lactamase (MBL). In this study, we discovered a novel hypothetical protein (HP) termed Bleg1_2437 from the genome of alkaliphilic Bacillus lehensis G1 which exhibited MBL-like properties of B3 subclass; but evolutionary divergent from other circulating B3 MBLs. Domain and sequence analysis of HP Bleg1_2437 revealed that it contains highly conserved Zn2+-binding residues such as H54, H56, D58, H59, H131 and H191, important for catalysis, similar with the subclass B3 of MBL. Built 3-D Bleg1_2437 structure exhibited an αββα sandwich layer similar to the well-conserved global topology of MBL superfamily. Other features include a ceiling and floor in the model which are important for accommodation and orientation of β-lactam antibiotics docked to the protein model showed interactions at varying degrees with residues in the binding pocket of Bleg1_2437. Hydrolysis activity towards several β-lactam antibiotics was proven through an in vitro assay using purified recombinant Bleg1_2437 protein. These findings highlight the presence of a clinically important and evolutionary divergent antibiotics-degrading enzyme within the pools of uncharacterized HPs.
The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.
A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.
A total of 58 Gram-positive bacteria strains were isolated from the marine environment and screened for potential probiotics for disease prevention and improving the productivity of tiger grouper Epinephelus fuscoguttatus larvae and juveniles. The bacteria were identified as Bacillus licheniformis, B. subtilis, B. circulans, B. sphaericus, B. cereus, Brevibacillus brevis, Corynebacterium propinquum, Leifsonia aquatica and Paenibacillus macerans. Only 24 strains showed antagonistic activities against four pathogenic strains; Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and Aeromonas hydrophila, where two of the Bacillus strains, B12 and B45 demonstrated intermediate to highest level of inhibitory activity against these pathogenic strains, respectively. Further assessment by co-culture assay showed that Bacillus strain B12 exhibited a total inhibition of V. alginolyticus, while B45 strain displayed no inhibitory activity. Mixed culture of Bacillus B12 and B45 strains to outcompete V. alginolyticus was observed at a cell density of 10(7) CFU ml(-1). Molecular identification and phylogenetic tree analysis have categorized Bacillus strain B12 to the reference strains GQ340480 and JX290193 of? B. amyloliquafaciens, and Bacillus strain B45 with a reference strain JF496522 of B. subtilis. Safety tests of probionts by intraperitoneal administration of B12 and B45 strains at cell densities of 103, 105 and 10(7) CFU ml(-1) revealed no abnormalities and cent percent survival for healthy Epinephelus fuscoguttatus juveniles within 15 days of experimental period. Overall, the study revealed that Bacillus B12 strain possesses tremendous probiotic potential that could be used as a feed supplement in tiger grouper diets. ?
Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.
The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.
A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.
Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.
A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.
Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide-protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.
Heavy metals from industrial effluents and sewage contribute to serious water pollution in most developing countries. The constant penetration and contamination of heavy metals into natural water sources may substantially raise the chances of human exposure to these metals through ingestion, inhalation, or skin contact, which could lead to liver damage, cancer, and other severe conditions in the long term. Biosurfactant as an efficient biological surface-active agent may provide an alternative solution for the removal of heavy metals from industrial wastes. Biosurfactants exhibit the properties of reducing surface and interfacial tension, stabilizing emulsions, promoting foaming, high selectivity, and specific activity at extreme temperatures, pH, and salinity, and the ability to be synthesized from renewable resources. This study aimed to produce biosurfactant from renewable feedstock, which is used cooking oil (UCO), by a local isolate, namely Bacillus sp. HIP3 for heavy metals removal. Bacillus sp. HIP3 is a Gram-positive isolate that gave the highest oil displacement area with the lowest surface tension, of 38 mN/m, after 7 days of culturing in mineral salt medium and 2% (v/v) UCO at a temperature of 30 °C and under agitation at 200 rpm. An extraction method, using chloroform:methanol (2:1) as the solvents, gave the highest biosurfactant yield, which was 9.5 g/L. High performance liquid chromatography (HPLC) analysis confirmed that the biosurfactant produced by Bacillus sp. HIP3 consists of a lipopeptide similar to standard surfactin. The biosurfactant was capable of removing 13.57%, 12.71%, 2.91%, 1.68%, and 0.7% of copper, lead, zinc, chromium, and cadmium, respectively, from artificially contaminated water, highlighting its potential for bioremediation.