Comparative Genome Analysis of a Novel Alkaliphilic Actinobacterial Species Nesterenkonia Haasae

In the present study, a comparative genome analysis of the novel alkaliphilic actinobacterial Nesterenkonia haasae with other members of the genus Nesterenkonia was performed. The genome size of Nesterenkonia members ranged from 2,188,008 to 3,676,111 bp. N. haasae and Nesterenkonia members of the present study encode the essential glycolysis and pentose phosphate pathway genes. In addition, some Nesterenkonia members encode the crucial genes for Entner-Doudoroff pathways. Some Nesterenkonia members possess the genes responsible for sulfate/thiosulfate transport system permease protein/ ATP-binding protein and conversion of sulfate to sulfite. Nesterenkonia members also encode the genes for assimilatory nitrate reduction, nitrite reductase, and the urea cycle. All Nesterenkonia members have the genes to overcome environmental stress and produce secondary metabolites. The present study helps to understand N. haasae and Nesterenkonia members’ environmental adaptation and niches specificity based on their specific metabolic properties. Further, based on genome analysis, we propose reclassifying Nesterenkonia jeotgali as a later heterotypic synonym of Nesterenkonia sandarakina.     

嗜碱芽孢杆菌产碱性纤维素酶研究概况

碱性纤维素酶是重要的洗涤剂添加剂,不仅可以去除衣物上的污渍,还可以软化衣物和增加衣物的鲜艳度。嗜碱芽孢杆菌是生产碱性纤维素酶的主要菌种。本文概述了其编码的碱性纤维素酶的性质,编码酶的基因、基因的克隆、表达以及作为洗涤剂添加剂的去污机理,指出了今后的研究方向。     

Alkaliphilic soil actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

Efficient Transfection of Novel Bovine Papillomavirus 1 Expression Plasmids

A series of novel bovine papillomavirus type 1 (BPV-1)-based expression plasmids was constructed and characterized in vitro as a starting point for the development of an in vivo gene therapeutic method. The order of transfection efficiency for different pBPVlacZ plasmids was pCGalBPV > pTKBPV > pSRalphaBPV in CV1-P cells. In the absence of selection pressure, the expression of pCGalBPVlacZ and pTKBPVlacZ was associated with long-term maintenance. In a comparison of pBPVlacZ with pSVlacZ, expression was maintained up to 12-17 and 8-12 days, respectively. The transfection of pBPVlacZ plasmids was efficient in secondary and primary, dividing and nondividing, neural and nonneural, and human cells and, furthermore, independent of the cell cycle as seen in growing as well as resting cells. All these characteristics are likely to be relevant for in vivo conditions, under which the percentage of proliferating cells could be quite low. In conclusion, the pBPV plasmids were efficiently delivered and expressed in different host cells, and therefore their performance in gene therapy is worth testing.     

Alkaliphilic Soil Actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

Identification of a Novel Alkaliphilic Esterase Active at Low Temperatures by Screening a Metagenomic Library from Antarctic Desert Soil

A novel esterase was identified through functional screening of a metagenomic library in Escherichia coli obtained from Antarctic desert soil. The 297-amino-acid sequence had only low (<29%) similarity to a putative esterase from Burkholderia xenovorans. The enzyme was active over a temperature range of 7 to 54°C and at alkaline pH levels and is a potential candidate for industrial application.The cold deserts of the McMurdo Dry Valleys, South Victoria Land, Eastern Antarctica, are widely acknowledged as having the harshest soil environments on Earth (6, 8, 26). Despite the apparent hostility of the environment, we and others have reported both unexpectedly high biomass (9) and phylogenetic diversity (1, 19, 24, 29) in Antarctic soils. The presence of numerous novel taxa suggests that these soils might prove to be valuable sources of genetic material for mining novel industrial enzymes active at low temperatures (9, 23).Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) catalyze the hydrolysis and synthesis of ester compounds. Their applications in industry cover a broad spectrum, including as detergent additives, in food processing, in environmental bioremediation, and in biomass and plant waste degradation for the production of useful organocompounds (3, 16).In this study, a novel esterase was isolated from a metagenomic fosmid library obtained from Antarctic soil. The enzyme displays activity over a wide temperature range and has very low similarity (<29% amino acid identity) to esterases in the GenBank database. The study demonstrates the value of Antarctic metagenomes as a source of novel industrial products.     

A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

Background

Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities.

Methodology/Principal Findings

Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220.

Conclusion

EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.     

Alkaline Anaerobic Respiration: Isolation and Characterization of a Novel Alkaliphilic and Metal-Reducing Bacterium

Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45°C (optimum, approximately 35°C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with Alkaliphilus transvaalensis and Alkaliphilus crotonatoxidans, respectively. The major phospholipid fatty acids were 14:1, 16:1ω7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name Alkaliphilus metalliredigens sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.     

嗜热脂肪芽孢杆菌质粒的分离

从堆肥和污泥中分离到一批抗药性高温细菌,经电泳检查,发现6株高温细菌细胞中有质粒存在。其中,嗜热脂肪芽孢杆菌T653的细胞DNA提取液电泳图谱上,有三条非染色体DNA条带,用电镜直接观察,证明它们是T653细胞中的三个质粒。测得两个较小质粒的分子量分别为3.6×10~6和45×10~6道尔顿。研究了嗜热脂肪芽孢杆的T653的温度生长条件与其细胞中质粒的关系。T653细胞中三个质粒的明确功能有待进一步探讨。     

Isolation and Characterization of Two Novel Plasmids from Pathogenic Leptospira interrogans Serogroup Canicola Serovar Canicola Strain Gui44

Background

Previous genomic analysis of pathogenic Leptospira has identified two circular chromosomes but no plasmid. This study aims to investigate potential extrachromosomal elements of L.interrogans serovar Canicola strain Gui44.

Methodology

Two novel plasmids, pGui1 and pGui2, were isolated from the pathogenic strain Gui44, using a modified alkaline lysis method. Southern blotting was performed to determine the presence and size of them. Then, 454 and Hiseq sequencing were applied to obtain and analyze the complete sequences of the two plasmids. Furthermore, real-time quantitative PCR and next-generation sequencing were used to compare relative copy numbers of the two plasmids with that of the chromosomes. Finally, after serial passages in vitro for more than 2 years, the strain Gui44 was subsequently re-sequenced to estimate stability of the two plasmids.

Principal Findings

The larger plasmid, pGui1, 74,981 base pairs (bp) in length with GC content of 34.63%, possesses 62 open reading frames (ORFs). The smaller plasmid, pGui2, is 66,851 bp in length with GC content of 33.33%, and contains 63 ORFs. The replication initiation proteins encoded by pGui1 and pGui2 demonstrate significant sequence similarity with LA1839 (86% and 88%), a well-known replication protein in another pathogenic L.interrogans serovar Lai strain Lai, suggesting the ability for autonomous plasmid replication. Quantitative PCR and next-generation sequencing confirms a single copy of both plasmids and their stable presence in the strain Gui44 with in vitro serial passages after more than 2 years. Interestingly, the two plasmids both contain a significant number of novel genes (35 in pGui1 and 52 in pGui2).

Conclusions

This report confirms the presence of two separate circular plasmids in serovar Canicola strain Gui44 and provides a new understanding of genomic organization, adaptation, evolution and pathogenesis of Leptospira, which will aid in the development of in vivo genetic manipulation systems in pathogenic Leptospira species.     

Plasmids from bacterial endosymbionts of hump-killer paramecia

Six isolates ofCaedibacter taeniospiralis, collected from four continents, were screened for plasmid DNA. Plasmid DNA species containing between 41.5 and 49.5 kilobase pairs (kb) were observed in all strains. Physical maps of plasmids were constructed by determining relative positions of the restriction endonuclease (BamHI,SalI,XhoI,SacI,PstI,AvaI, andEcoRI) recognition sequences in each plasmid. The physical map of the smallest plasmid (41.5 kb), pKAP30, is reflected in each of the plasmids isolated from the other strains ofC. taeniospiralis. Plasmid DNA from three of the isolates (strains 51 and 116 both from Indiana and strain 169 from Japan) each contain 43 kb, where 41.5 kb appear to be identical to pKAP30 (obtained from the Australian strain, A30). The extra 1.5 kb present in pKAP51, pKAP116, and pKAP169 is included as a single polynucleotide sequence. The 1.5-kb inclusion is located at apparently identical positions in pKAP116 and pKAP169 and at a totally different position in pKAP51. The two remaining plasmids, pKAP47 (from California strain 47) and pKAP298 (from Panama strain 298), both contain 49 kb to include a continuous 41.5-kb sequence that is apparently identical to pKAP30. The results indicate that the polynucleotide sequences of these plasmids are highly conserved and that the observed variations among them may be accounted for by transposable elements.     

Isolation and Characterization of Halotolerant Alkaliphilic Methanotrophic Bacteria from Tuva Soda Lakes

Two strains (5Z and 20Z) of halotolerant alkaliphilic obligate methanotrophic bacteria were first isolated from moderately saline soda lakes in Tuva (Central Asia). The strains grow fastest at pH 9.0–9.5 and much more slowly at pH 7.0. No growth occurred at pH ≤ 6.8. They require NaHCO₃ or NaCl for growth in alkaline medium. Gram-negative, motile rods with ordered cup-shaped cell wall structures and Type I intracytoplasmic membranes assimilate methane and methanol via the ribulose monophosphate pathway. The G + C content of strains 5Z and 20Z are 47.6 and 47.9 mol%, respectively. Based on their alkaliphilic physiology, both strains were referred to as Methylobacter alcaliphilus sp. nov. The changes in cell phospholipids, fatty acids, and amino acids have been observed upon varying salinity and pH of the medium, thus suggesting structure-function osmoadaptation of the strains studied. Whole-cell experiments revealed the salt- and pH-dependence of CH₄ oxidation and assimilation rates. Cell motility was also Na⁺ dependent and sensitive to some energy uncouplers and ionophores. Received: 7 March 1997 / Accepted: 14 April 1997     

Alkaliphilic and halophilic hydrocarbon-utilizing bacteria from Kuwaiti coasts of the Arabian Gulf

Green animate materials from the intertidal zone of the Arabian Gulf coast accommodated more alkaliphilic and halophilic bacteria than inanimate materials. The alkaliphilic oil-utilizing bacteria, as identified by their 16S ribonucleic acid sequences, belonged to the following genera arranged in decreasing frequences: Marinobacter, Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus. The halophilic oil-utilizing bacteria belonged to the genera: Marinobacter, Georgenia, Microbacterium, Stappia, Bacillus, Isoptericola, and Cellulomonas. Most isolates could grow on a wide range of pure n-alkanes and aromatic compounds, as sole sources of carbon and energy. Quantitative gas liquid chromatographic analysis showed that individual isolates attenuated crude oil and representative pure hydrocarbons in culture. The optimum pH for most of the alkaliphilic genera was pH 10, and the optimum salinity for the halophiles ranged between 2.5 and 5% NaCl (w/v). It was concluded that as far as their microbial makeup is concerned, oily alkaline and saline intertidal areas of the Kuwaiti coasts have a self-cleaning potential.     

Plasmids from bacilli related to BAcillus subtilis]

The basic structural and functional properties of natural bacilli plasmids are analyzed in this review. Bacilli plasmids are mostly cryptic, but some are found to have selective markers. Small plasmids replicate by rolling-circle mechanism, however, the replication of large plasmids is likely to occur by the theta-mechanism. Plasmid structures involved in replication are analyzed. The bacilli plasmids are stable. They are promising material for vector construction.     

Novel Method of Single Antibiotic Selection of Cells Containing Multiple Stage-Specific Promoter-Fluorophore Plasmids

This study demonstrates a novel method by which multiple separate plasmids can be stably integrated into the genome using single antibiotic resistance for selection. This method was used to integrate three different cardiac-specific promoters driving different fluorophores into murine embryonic stem cells, allowing sequential visualization of various stages of cardiac differentiation. This method is broadly applicable to the study of cell lineage of different stem/progenitor cells.     

Functional Analysis of Three Plasmids from Lactobacillus plantarum

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.     

Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein⁻¹, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)₈ barrel motif in domain A.     

Novel α-Amylase That Is Highly Resistant to Chelating Reagents and Chemical Oxidants from the Alkaliphilic Bacillus Isolate KSM-K38

A novel α-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60°C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H₂O₂ (1.8 M).     

Acquisition of Iron by Alkaliphilic Bacillus Species

The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknown. We demonstrate that alkaliphilic (pH > 9) Bacillus species are sensitive to artificial iron (Fe³⁺) chelators and produce iron-chelating molecules. These alkaliphilic siderophores contain catechol and hydroxamate moieties, and their synthesis is stimulated by manganese(II) salts and suppressed by FeCl₃ addition. Purification and mass spectrometric characterization of the siderophore produced by Caldalkalibacillus thermarum failed to identify any matches to previously observed fragmentation spectra of known siderophores, suggesting a novel structure.Iron is an abundant element in nature; however, in most aqueous aerobic environments iron forms insoluble ferric hydroxide, Fe(OH)₃. This poses a major problem for most aerobic bacteria, as ferric hydroxide has a solubility constant of 10⁻³⁹ M, therefore limiting the concentration of ferric ions to 10⁻¹⁸ M at pH 7.0. For example, bacteria living in seawater (approximate pH 8.0) require iron, yet dissolved iron is only present at 0.02 to 2.0 nM (5). Despite this apparent lack of bioavailability, iron has been repeatedly demonstrated to be an essential element for aerobic bacterial growth (1).With the lack of readily accessible iron at physiological pH, most bacteria have evolved systems to deal with the incumbent problem of iron acquisition. Under iron-rich conditions, Fe²⁺ uptake receptors, such as FeoAB, are synthesized in bacteria, which passively import iron in the immediate vicinity of the cell (1, 23). No equivalent system has been identified for Fe³⁺ transport. To acquire Fe³⁺ under aqueous aerobic conditions, bacteria commonly have import systems involving the synthesis, secretion, and regathering of a group of secondary metabolites known as siderophores (1, 11). Siderophores are low-molecular-weight chemical moieties that chelate Fe³⁺ and typically have complex formation (K_f) constants in the range of 10²³ to 10⁵² (11). Siderophores, like other chelators, are known to increase the solubility of iron by hindering the formation of Fe-oxyhydroxides at high pH, at which the Fe-oxyhydroxides are the dominating inorganic species (27). Siderophores are also known to facilitate the dissolution of Fe from minerals (3). Siderophore-iron complexes can either be transported through cellular membranes using dedicated transport systems or if the Fe(III) central atom is reduced, making the iron bioavailable for cellular processes (10, 14). Three major groups of siderophores have been described in bacteria: hydroxamates, catecholates, and carboxylates. Hydroxamates and catechols are commonly produced by aerobic bacteria living at neutral to alkaline pH, whereas carboxylates are significantly more common in bacteria living in mildly acidic pH (11-13). In the genus Bacillus, Bacillus megaterium and Bacillus subtilis are producers of schizokinen and bacillibactin, respectively (6, 20). Bacillus anthracis produces both a catechol and a hydroxamate siderophore (7, 34), and B. licheniformis strain VK21 is the only known example of a thermoresistant catecholate-producing Gram-positive bacterium (32).Although there is extensive literature on iron capture mechanisms in bacteria that thrive at neutral pH, there is little information at a biochemical or molecular level on how aerobic bacteria growing at extreme alkaline pHs (i.e., pH 9 to 11) acquire iron. At alkaline pH, the solubility constant for iron decreases far below the requirement for living cells, and the concentration of bioavailable iron is estimated to be approximately 10⁻²³ M at pH 10 (11). Taking this extreme lack of iron into account, the sequestering mechanisms of alkaliphilic bacteria must be powerful, yet there has been little analysis of the types of iron-chelating molecules these bacteria produce.