Biosorption of Copper by Paenibacillus polymyxa Cells and their Exopolysaccharide

Summary Biosorption of heavy metals by gram-positive, non-pathogenic and non-toxicogenic Paenibacillus polymyxa P13 was evaluated. Copper was chosen as a model element because it is a pollutant originated from several industries. An EPS (exopolysaccharide)-producing phenotype exhibited significant Cu(II) biosorption capacity. Under optimal assay conditions (pH 6 and 25 °C), the adsorption isotherm for Cu(II) in aqueous solutions obeyed the Langmuir model. A high q value (biosorption capacity) was observed with whole cells (q_max=112 mgCu g⁻¹). EPS production was associated with hyperosmotic stress by high salt (1 M NaCl), which led to a significant increase in the biosorption capacity of whole cells (q_max=150 mgCu g⁻¹). Biosorption capacity for Cu(II) of the purified EPS was investigated. The maximum biosorption value (q) of 1602 mg g⁻¹ observed with purified EPS at 0.1 mg ml⁻¹ was particularly promising for use in field applications.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

An integrated high throughput strategy to screen mutants of Paenibacillus polymyxa with high polymyxin E-productivity

An integrated high-throughput screening (HTS) strategy was developed to screen large numbers of polymyxin E-producing mutants of Paenibacillus polymyxa. Various types of mutants were transferred onto the surfaces of solidified agar in 96-well microtiter plates, and then inoculated to 96-deep-well microtiter plates for micro-cultivation. The culture conditions were optimized for the production of polymyxin E. The supernatants from the micro-culture plates were transferred to 96-well bioassay microtiter plates containing Escherichia coli JM109 for high-throughput bioassay. By using this high-throughput screening (HTS) procedure, one best producer P. polymyxa PE 5.808 was identified from a large NTG mutated library with about 5,000 isolates. The volumetric productivity of polymyxin E of P. polymyxa PE 5.808 was 1,200 μg/ml in shake flasks, about 140% improvement compared with that of the wild type strain.     

Identification and functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681

Fusaricidin, a peptide antibiotic consisting of six amino acids, has been identified as a potential antifungal agent from Paenibacillus polymyxa. Here, we report the complete sequence of the fusaricidin synthetase gene (fusA) identified from the genome sequence of a rhizobacterium, P. polymyxa E681. The gene encodes a polypeptide consisting of six modules in a single open-reading frame. Interestingly, module six of FusA does not contain an epimerization domain, which suggests that the sixth amino acids of the fusaricidin analogs produced by P. polymyxa E681 may exist as an l-form, although all reported fusaricidins contain d-form alanines in their sixth amino acid residues. Alternatively, the sixth adenylation domain of the FusA may directly recognize the d-form alanine. The inactivation of fusA led to the complete loss of antifungal activity against Fusarium oxysporum. LC/MS analysis confirmed the incapability of fusaricidin production in the fusA mutant strain, thus demonstrating that fusA is involved in fusaricidin biosynthesis. Our findings suggested that FusA can produce more than one kind of fusaricidin, as various forms of fusaricidins were identified from P. polymyxa E681.     

Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.     

Enhancement of (R,R)-2,3-butanediol production from xylose by Paenibacillus polymyxa at elevated temperatures

Conversion of xylose to (R,R)-2,3-butanediol by Paenibacillus polymyxa in anaerobic batch and continuous cultures was increased by 39% and 52%, respectively, by increasing the growth temperatures from 30 to 39 °C. There was no effect of temperature when glucose was used as substrate. 39 mM (R,R)-2,3-butanediol, 65 mM ethanol, and 47 mM acetate were obtained from 100 mM xylose after 24 h batch culture at 39 °C. With 100 mM glucose and 100 mM xylose used together in a batch culture at 39 °C, all xylose was consumed after 24 h and 82 mM (R,R)-2,3-butanediol, 124 mM ethanol and 33 mM acetate were produced.     

Identification of Bacillus azotofixans using API tests

The identification of Bacillus azotofixans strains using API tests is described. Twenty-two strains were studied according to their fermentation pattern on 49 different carbohydrates. A profile of the B. azotofixans type strain is presented, together with an average profile of all strains tested. The fermentation pattern for B. azotofixans is also compared to those of the closely similar species B. polymyxa and B. macerans. These profiles may be useful for the identification of new strains.     

Identification of LI-F type antibiotics and di-n-butyl phthalate produced by Paenibacillus polymyxa

Paenibacillus polymyxa strain JSa-9, a soil isolate that displayed antibacterial and antifungal activities in vitro, had been found to produce two types of antimicrobial substances. The two compounds were extracted from the fermentation broth of JSa-9 using ethyl acetate and subsequently purified by high performance liquid chromatography. By means of liquid chromatography-mass spectrometry and tandem mass spectrometry analysis, one of two antagonistic compounds was determined as di-n-butyl phthalate. And another was characterized as a mixture of related peptides of molecular masses of 883, 897, 911, 947, and 961 Da, with the most likely structure of them determined to be a cyclic depsipeptide with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety bound to a free amino group. These peptides were therefore members of the LI-F group of cyclic depsipeptides.     

Optimization, purification, characterization and antioxidant activity of an extracellular polysaccharide produced by Paenibacillus polymyxa SQR-21

The optimization, purification and characterization of an extracellular polysaccharide (EPS) from a bacterium Paenibacillus polymyxa SQR-21 (SQR-21) were investigated. The results showed that SQR-21 produced one kind of EPS having molecular weight of 8.96 × 10⁵ Da. The EPS was comprised of mannose, galactose and glucose in a ratio of 1.23:1.14:1. The ratio of monosaccharides and glucuronic acid was 7.5:1. The preferable culture conditions for EPS production were pH 6.5, temperature 30 °C for 96 h with yeast extract and galactose as best N and C sources, respectively. The maximum EPS production (3.44 g L⁻¹) was achieved with galactose 48.5 g L⁻¹, Fe³⁺ 242 μM and Ca²⁺ 441 μM. In addition, the EPS showed good superoxide scavenging, flocculating and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities were determined. These results showed the great potential of EPS produced by SQR-21 to be used in industry in place of synthetic compounds.     

Ecology and biotechnological potential of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis>: a minireview

Microbial diversity is a major resource for biotechnological products and processes. Bacteria are the most dominant group of this diversity which produce a wide range of products of industrial significance. Paenibacillus polymyxa (formerly Bacillus polymyxa), a non pathogenic and endospore-forming Bacillus, is one of the most industrially significant facultative anaerobic bacterium. It occurs naturally in soil, rhizosphere and roots of crop plants and in marine sediments. During the last two decades, there has been a growing interest for their ecological and biotechnological importance, despite their limited genomic information. P. polymyxa has a wide range of properties, including nitrogen fixation, plant growth promotion, soil phosphorus solubilisation and production of exopolysaccharides, hydrolytic enzymes, antibiotics, cytokinin. It also helps in bioflocculation and in the enhancement of soil porosity. In addition, it is known to produce optically active 2,3-butanediol (BDL), a potentially valuable chemical compound from a variety of carbohydrates. The present review article aims to provide an overview of the various roles that these microorganisms play in the environment and their biotechnological potential.     

Isolation and characterization of peptide antibiotics LI-F04 and polymyxin B6 produced by Paenibacillus polymyxa strain JSa-9

Paenibacillus polymyxa JSa-9 had been found to produce five cyclic LI-F type antibiotics which were released into culture medium in accordance with our previous report. In this study, another three kinds of antagonistic compounds were extracted from P. polymyxa JSa-9 cell pellets and (or) spores by methanol. Using high performance liquid chromatography (HPLC) method, two antagonistic fractions were separated and collected from the methanol extract. One showed inhibition against Escherichia coli and Staphylococcus aureus, while the other was active against Aspergillus niger and S. aureus. By means of electrospray ionization mass spectroscopy (ESI-MS), infrared spectroscopy (IR), and amino acid analysis, two kinds of compounds from fraction B with molecular masses of 901 and 915 Da were characterized as the linear lipopeptide analogs of antibiotics LI-F04a and LI-F04b, respectively. Another antimicrobial substance from fraction A could be attributed to polymyxin B₆.     

Evaluation of Biolog system for identification of strains of Paenibacillus azotofixans

Biolog system was evaluated for the identification of strains of Paenibacillus azotofixans as no data concerning this species were in the list of Bacillus currently identified using Biolog data base. The P. azotofixans type strain P3L5 was first tested with the results recorded manually or using the automatic plate reader. In both cases, P3L5 utilized 22 carbon sources and when the results obtained were compared to data of Biolog software (Release 3.50), P3L5 was identified as B. azotoformans with a similarity coefficient of 0.913 (data recorded manually) and of 0.791 (data recorded automatically). Metabolic profiles of P3L5 were also compared after readings of 4 and 24 h using the computer-driven automatic plate reader. No significant difference was observed in both cases and P3L5 was identified again as B. azotoformans with indices of similarity considered only for excellent identification. Besides P3L5, other 15 P. azotofixans strains were tested with the Biolog system and all were identified as B. azotoformans with similarity coefficients varying from 0.511 to 0.927. Phenotypic and genetic characteristics of B. azotoformans were compared to those described for P. azotofixans to explain the misidentification of the latter species. We could conclude that these two species are quite different and that data of Biolog software are from P. azotofixans and not from B. azotoformans.     

Auxin production and detection of the gene coding for the Auxin Efflux Carrier (AEC) protein in <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis>

Different species of Paenibacillus are considered to be plant growth-promoting rhizobacteria (PGPR) due to their ability to repress soil borne pathogens, fix atmospheric nitrogen, induce plant resistance to diseases and/or produce plant growth-regulating substances such as auxins. Although it is known that indole-3-acetic acid (IAA) is the primary naturally occurring auxin excreted by Paenibacillus species, its transport mechanisms (auxin efflux carriers) have not yet been characterized. In this study, the auxin production of P. polymyxa and P. graminis, which are prevalent in the rhizospheres of maize and sorghum sown in Brazil, was evaluated. In addition, the gene encoding the Auxin Efflux Carrier (AEC) protein from P. polymyxa DSM36(T) was sequenced and used to determine if various strains of P. polymyxa and P. graminis possessed this gene. Each of the 68 P. polymyxa strains evaluated in this study was able to produce IAA, which was produced at concentrations varying from 1 to 17 microg/ml. However, auxin production was not detected in any of the 13 P. graminis strains tested in this study. Different primers were designed for the PCR amplification of the gene coding for the AEC in P. polymyxa, and the predicted protein of 319 aa was homologous to AEC from Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis. However, no product was observed when these primers were used to amplify the genomic DNA of seven strains of P. graminis, which suggests that this gene is not present in this species. Moreover, none of the P. graminis genomes tested were homologous to the gene coding for AEC, whereas all of the P. polymyxa genomes evaluated were. This is the first study to demonstrate that the AEC protein is present in P. polymyxa genome.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Construction of a new vector for the expression of foreign genes inZymomonas mobilis

Summary Broad host range plasmids have previously been shown to be suitable as vectors to introduce antibiotic resistance genes intoZ. mobilis. However, attempts to use these vectors to carry other genes with enteric promoters and controlling elements have resulted in limited success due to poor expression. Thus we have constructed a promoter cloning vector in a modified pBR327 and used this vector to isolated 12 promoters fromZ. mobilis which express various levels of -galactosidase inEscherichia coli. Four of these were then subcloned into pCVD 305 for introduction intoZ. mobilis. All expressed -galactosidase inZ. mobilis with activities of 100 to 1800 Miller units. One of these retained aBamHl site into which new genes can be readily inserted immediately downstream from theZ. mobilis promoter. Genetic traits carried by pCVD 305 were initially unstable but spontaneous variants were produced during sub-culture in which the plasmid was resistant to curing at elevated temperature. One of these variants was examined in some detail. The increased stability of this variant appears to result from an alteration in the plasmid rather than a chromosomal mutation or from chromosomal integration.     

Inoculationin vitro of wheat seedlings and wheat straw cultures withBacillus azotofixans

Bacillus azotofixans is a recently described species capable of fixing molecular nitrogen efficiently.Ecological studies performed in monoxenic wheat cultures, both in 0.7% agar and in vermiculite-sand mixture, showed that no acetylene reduction occurred and that this bacteria did not grow when supplied only with the wheat plant root exudates. However, after glucose addition to the 0.7% agar cultures, acetylene reduction ability (ARA) was detected. Comparing ARA for media with glucose both with and without plants, it was observed that the plants supply some component leading to the increase of the nitrogenase activity, since the ARA doubled in the samples containing plants.In wheat straw cultures a fast growth of the bacteria was observed in the first 24 hours after inoculation, but no acetylene reduction was detected. After glucose addition to the media with and without straw, nitrogenase activity was detected.     

Composition and immunochemical characteristics of exopolysaccharides from the rhizobacterium <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> 1465

Exopolysaccharides (EPS) synthesized by Paenibacillus polymyxa 1465 in the course of batch cultivation were proven to contain neutral and acidic fractions. EPS are heterogeneous polysaccharides, represented by a complex of macromolecules with molecular mass of 7 × 10⁴ to 2 × 10⁶ Da. The acidic component was shown to be predominant in EPS preparations isolated from bacteria cultivated on glucose, which corresponds to a higher viscosity of EPS water solutions. The exoglycans were shown to contain glucose, mannose, galactose, and uronic acids. Polyclonal rabbit antibodies against the isolated P. polymyxa 1465 EPS preparations were used in a comparative immunodiffusion analysis of a number of P. polymyxa strains.     

Cloning and characterization of an exoinulinase from Bacillus polymyxa

A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced. It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55522 Da. Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified. The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2. Inulinase activity was optimal at 35°C and pH 7, was completely inactivated by 1 mM Ag⁺ or Hg²⁺. The K _m and V _max values for inulin hydrolysis were 0.7 mM and 2500 M min^–1 mg^–1 protein. The enzyme acted on inulin via an exo-attack to produce fructose mainly.     

Production of a potentially novel anti-microbial substance by Bacillus polymyxa

A bacterial strain, SCE2, identified as Bacillus polymyxa, produced an anti-microbial substance active against yeasts, fungi and different genera of Gram-positive and-negative bacteria, in liquid medium and in plate assays. This substance appeared to be an antibiotic different from the polymyxin group, mainly because of its action against the majority of Gram-positive bacteria tested and its lack of activity against Pseudomonas aeruginosa, a species usually killed by polymyxins. Preliminary characterization showed resistance to heat (65°C, 2 h), to proteases, trypsin, lysozyme, deoxyribonuclease I, ribonuclease A, phospholipase C, ethanol, acetone, chloroform, ether and to strong alkali treatment (2 M NaOH). The molecular weight was less than 3500. The B. polymyxa strain harboured a plasmid that did not correlate with antibiotic production; after curing experiments, a derivative strain, SCE2(46), was isolated that lacked the plasmid pES1, but showed the same inhibitory spectrum as the wild-type strain.