Essay: Isolation and characteristics of a novel thermophilic bacterium from soil

Abstract

We describe the isolation and characteristics of a novel thermophilic bacterium from soil. The organism is a member of the Anoxybacillus genus based on phylogenetic analysis of the 16S rRNA gene. The 16S rRNA of the organism shares >99% sequence identity with those of two species, A. rupiensis and A. geothermalis. We named this isolate as Anoxybacillus sp. strain UARK-01. UARK-01 grows optimally in the presence of oxygen at 55C and pH 8. It grew excellently in the presence of lignin as the sole carbon source. Culture supernatant from UARK-01 grown on lignin was rich in laccase activity. The laccase activity was optimal at 90°C and pH 9, and there was comparable activity at 80C and 100C. The crude laccase decolorized approximately 75% of Congo Red in 7 hours under optimal conditions. A single laccase gene was identified from the draft genome sequence of Anoxybacillus sp. UARK-01. The UARK-01 laccase (Anox_Lacc) was cloned and overexpressed in Escherchia coli and was partially purified. The partially purified Anox_Lacc decolorized approximately 1.64+/0.21 nanomoles of Congo Red per microgram protein in 30 minutes at 90C and pH 9. Anox_Lacc is a member of the multi-copper polyphenol oxidoreductase laccase family (pfam02578 Cu-oxidase_4) and has novel characteristics. Multiple sequence analysis of Anox_Lacc with six homologs from the family revealed four conserved copper ligands and several new residues that are fully conserved. Anox_Lacc is enriched in leucine, glutamine and lysine, and it contains fewer alanine, arginine, glycine, and serine residues. Skewed amino acid composition of Anox_Lacc likely contributes to the exceptional thermochemical properties of the laccase activity from UARK-01. Both lignin utilization and production of hyperthermostable alkaline laccase are new findings in the Anoxybacillus genus.

2.1 INTRODUCTION

Laccases (EC 1.10.3.2) are multicopper-containing polyphenol oxidases that oxidize variety of substrates including phenols, polyphenols, amino phenols, methoxy phenols, and aromatic amines [17]. There is an ever-increasing demand for thermostable laccases for industrial processes and environmental bioremediation [16, 28]. One of the major applications of laccases includes the decolorization of dyes in textile effluents [25]. Every year up to 200,000 tons of dyes are released in effluents from textile industry. Toxic and mutagenic dyes in the effluents pose serious environmental and health concerns [25, 28]. Thermostable microbial laccases can be employed to effectively remove dyes from contaminated water [35]. Another important application for laccases includes the delignification of lignocellulose [10, 19]. Lignocellulose is the most abundant renewable resource for sustainable production of bioenergy and value-added chemicals [22]. However, the polyphenolic lignin component of lignocellulose is a major barrier in its bioconversion to valuable products [31]. Thermostable laccases can be utilized to effectively pretreat lignocellulose and remove phenolic inhibitors [10, 18, 31]. While laccases are produced by plants, fungi and bacteria, most plant and fungal laccases are not very thermostable [16].

The genus Anoxybacillus is a relatively new genus of Gram-positive bacteria that was proposed in the year 2000 [24]. It is most closely related to the genus Geobacillus and belongs to the family Bacillaceae. The genus name, Anoxybacillus, which was derived from its first described aerotolerant-anaerobic species, suggests that these organisms are anaerobic. However, mostly facultative anaerobic and strict aerobic members have been described since the first introduction of the genus [15]. To date, the genus is composed of 22 species and two subspecies. Anoxybacillus members are moderately thermophilic with optimum growth temperature (Topt) around 50°C−60°C, and their optimum pH (pHopt) for growth can range from 5.5 to 9.7 [15]. Emerging data from biochemical and genomic studies are reinforcing the importance of Anoxybacillus genus as a potential new resource for thermostable enzymes. While starch, cellulose, xylan, and arabinofuran degradation capabilities have been identified in different Anoxybacillus species, lignin degradation capabilities remain to be studied in this genus [15]. Here, we describe the isolation and characteristics of Anoxybacillus sp. strain UARK-01 (referred to as UARK-01 below), a new thermophilic bacterium that utilizes lignin as a carbon source and produces hyperthermostable alkaline laccase activity.

2.2 MATERIALS AND METHODS

2.2.1 Isolation of microorganisms

In a search for new thermophilic lignocellulose-degrading microorganisms from soil, we used isolation media containing switchgrass as the sole carbon source. Isolation media contained mineral salts (per liter: 1.695 g Na2HPO4, 0.75 g KH2PO4, 0.125 g NaCl, and 0.25 g NH4Cl) and 0.5% (w/v) switchgrass as the sole carbon source. Solid media was prepared by adding 2% agar (BD Difco); all media were sterilized using autoclave. Soil sample collected from 3 inches below a grass lawn on the university campus was inoculated into isolation media and incubated at 50C. After regular intervals, small aliquots were spread on solid media plates and incubated at 50C until microbial colonies developed. From the colonies, microorganisms were isolated into pure cultures through extensive successive culturing of single colonies on solid media plates. Phase contrast microscopy was used to also assess the segregation of microorganisms into pure cultures.

2.2.2 Media and culture conditions

UARK-01 was routinely cultured in mineral salts media (composition described above) at pH 8.0, 55°C and 250 rpm. Depending on the experiment, sole carbon source was provided in the form of either yeast extract, peptone, glucose, cellobiose, xylose, cellulose, carboxymethyl cellulose, beechwood xylan, or alkali lignin (all chemicals were purchased from Sigma-Aldrich). Prior to use, lignin, cellulose, and agar were washed extensively with deionized sterile water to remove any soluble contaminants. For determining optimum pH for growth, yeast extract (0.5% w/v) was used as the sole carbon source and the pH of the medium was adjusted using either sodium hydroxide or phosphoric acid. Bacterial growth in liquid media was measured using optical density at 600 nm.

2.2.3 Bacterial identification

The 16S rRNA gene was amplified from the culture lysate of UARK-01, using polymerase chain reaction (PCR) and two bacteria-specific primers 8F and 1492R [14]. The PCR product was analyzed using agarose gel electrophoresis and DNA was purified from the gel using QIAquick Gel Extraction Kit (Qiagen). Purified DNA was sequenced from both ends at the DNA Resource Center on campus. The 16S rRNA gene sequence of UARK-01 was searched against the NCBI nr database using BLAST [1].

2.2.4 16S phylogenetic analysis

Nucleotide sequences of 16S rRNA genes from twenty-four bacteria were obtained from the GenBank database and were aligned with the 16S rRNA gene sequence of UARK-01. In order to make robust inferences, sequences were aligned separately using two independent programs, ClustalX and MUSCLE [9, 20]. Neighbor-joining trees were generated using ClustalX, and maximum-likelihood trees were generated using RAxML [30]. Non-parametric bootstrapping was performed with 1000 iterations and statistical bootstrap values were used to infer tree topology. Trees were visualized and annotated using the FigTree v1.4.2 software (http://tree.bio.ed.ac.uk/software/figtree/). The following 16S sequences were used for multiple sequence alignment and phylogenetic tree construction. A. geothermalis strain ATCC BBA-2555 (accession# KJ722458.1), A. rupiensis strain R270 (NR_042379.1), A. tepidamans strain GS5-97 (NR_025819.1), A. kualawohkensis strain ET10/3 (KJ722465.1), A. eryuanensis strain E-112 (NR_117229.1), A. mongoliensis strain T4 (NR_116097.1), A. kamchatkensis strain JW/VK-KG4 (NR_041915.1), A. salavatliensis strain A343 (NR_104492.1), A. tengchongensis strain T-11 (NR_116668.1), A. ayderensis strain AB04 (NR_024837.1), A. flavithermus strain DSM 2641 (NR_026516.1), A. contaminans strain R-16222 (NR_029006.1), A. amylolyticus strain MR3C (NR_042225.1), A. caldiproteolyticus strain R-35652 (NR_116989.1), A. voinovskiensis strain IHB B 4422 (KF475881.1), Anoxybacillus sp. SK3-4 (GQ184213.1), Anoxybacillus sp. DT3-1 (GU129931.1), A. gonensis strain O9 (KM596794.1), A. suryakundensis strain JS1 (KC958552.1), A. thermarum strain A4 (KC310455.1), Anoxybacillus sp. P3H1B (NZ_LPUG01000026.1 contig000026 region 1-1460), Anoxybacillus sp. BCO1 (NZ_JRLC01000086.1 contig000086 region 11-1554), Anoxybacillus sp. KU2-6(11) (NZ_JPZN01000047.1 contig58 region 337-1884), and Geobacillus thermodenitrificans strain BGSC W9A38 (AY608964.1). The 16S rRNA gene from G. thermodenitrificans was used as the outgroup.