Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01

We isolated the feather-degrading Bacillus pseudofirmus FA30-01 from the soil sample of poultry farm. The isolate completely degraded feather pieces after liquid culture at 30°C (pH 10.5) for 3 days. Strain FA30-01 is a Gram-positive, spore-forming, rod-shaped bacterium and was identified with B. pseudofirmus based on 16S rDNA analysis. The keratinase enzyme produced by strain FA30-01 was refined using ammonium sulfate precipitation, negative-ion DEAE Toyopearl exchange chromatography, and hydroxyapatite chromatography. The refinement level was 14.5-fold. The molecular weight of this enzyme was 27.5 kDa and it had an isoelectric point of 5.9. The enzyme exhibited activity at pH 5.1–11.5 and 30–80°C with azokeratin as a substrate, although the optimum pH and temperature for keratinase activity were pH 8.8–10.3 and 60°C, respectively. This enzyme is one of the serine-type proteases. Subtilisin ALP I and this enzyme had 90% homology in the N-terminal amino acid sequence. Since this enzyme differed from ALP I in molecular weight, heat resistance and isoelectric point, they are suggested to be different enzymes.     

Bacterial diversity and carbonate precipitation in the giant microbialites from the highly alkaline Lake Van, Turkey

Lake Van harbors the largest known microbialites on Earth. The surface of these huge carbonate pinnacles is covered by coccoid cyanobacteria whereas their central axis is occupied by a channel through which neutral, relatively Ca-enriched, groundwater flows into highly alkaline (pH ~9.7) Ca-poor lake water. Previous microscopy observations showed the presence of aragonite globules composed by rounded nanostructures of uncertain origin that resemble similar bodies found in some meteorites. Here, we have carried out fine-scale mineralogical and microbial diversity analyses from surface and internal microbialite samples. Electron transmission microscopy revealed that the nanostructures correspond to rounded aragonite nanoprecipitates. A progressive mineralization of cells by the deposition of nanoprecipitates on their surface was observed from external towards internal microbialite areas. Molecular diversity studies based on 16S rDNA amplification revealed the presence of bacterial lineages affiliated to the Alpha-, Beta- and Gammaproteobacteria, the Cyanobacteria, the Cytophaga-Flexibacter-Bacteroides (CFB) group, the Actinobacteria and the Firmicutes. Cyanobacteria and CFB members were only detected in surface layers. The most abundant and diverse lineages were the Firmicutes (low GC Gram positives). To the exclusion of cyanobacteria, the closest cultivated members to the Lake Van phylotypes were most frequently alkaliphilic and/or heterotrophic bacteria able to degrade complex organics. These heterotrophic bacteria may play a crucial role in the formation of Lake Van microbialites by locally promoting carbonate precipitation.     

<Emphasis Type="Italic">Bacillus</Emphasis> sp. WW3-SN6, a novel facultatively alkaliphilic bacterium isolated from the washwaters of edible olives

A novel Gram-positive facultatively alkaliphilic, sporulating, rod-shaped bacterium, designated as WW3-SN6, has been isolated from the alkaline washwaters derived from the preparation of edible olives. The bacterium is nonmotile, and flagella are not observed. It is oxidase positive and catalase negative. The facultative alkaliphile grows from pH 7.0 to 10.5, with a broad optimum from pH 8.0 to 9.0. It could grow in up to 15% (w/v) NaCl, and over the temperature range from 4° to 37°C, with an optimum between 27° and 32°C: therefore, it is both halotolerant and psychrotolerant. The bacterium is sensitive to a range of β-lactam, sulfonamide, and aminoglycoside antibiotics, but resistant to trimethoprim. The range of amino acids, sugars, and polyols utilized as growth substrates indicates that this alkaliphile is a heterotrophic bacterium. d(+)-glucose, d(+)-glucose-6-phosphate, d(+)-cellobiose, starch, or sucrose are the substrates best utilized. The major membrane lipids are phosphatidylglycerol and diphosphatidylglycerol, with smaller amounts of phosphatidylethanolamine and an unknown phospholipid. During growth at high pH, the proportion of phosphatidylglycerol is increased relative to phosphatidylethanolamine. The fatty acyl components in the membrane phospholipids are mainly branched chain, with 13-methyl tetradecanoic and 12-methyl tetradecanoic acids as the predominant components. The G + C content of the genomic DNA is 41.1 ± 1.0 mol%. The results of 16S ribosomal RNA sequence analysis place this alkaliphilic bacterium in a cluster, together with an unnamed alkaliphilic Bacillus species (98.2% similarity). Received: October 22, 1999 / Accepted: February 27, 2000     

Salinivibrio costicola subsp. alcaliphilus subsp. nov., a haloalkaliphilic aerobe from Campania Region (Italy)

Phenotypic and phylogenetic studies were performed on unidentified Gram-negative staining, haloalkaliphilic aerobe and protease producer Salinivibrio-like organism recovered from a saltish spring with algal mat in the “Pozzo del Sale” site (Salt's Well) in the Campania Region (South Italy). Phylogenetic analysis based on comparison of 16S rRNA gene sequences demonstrated that the isolate was related to species of Salinivibrio genus. The DNA–DNA hybridization of the type strain 18AG^T with the most related Salinivibrio costicola subsp. costicola showed a re-association value of 72%. Based on the phenotypic distinctiveness of 18AG^T strain and molecular, chemical and genetic evidence, it is proposed that strain 18AG^T can be classified as S. costicola subsp. alcaliphilus, subsp. nov. The type strain of S. costicola subsp. alcaliphilus, is ATCC BAA-952^T; DSM 16359^T.     

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detegents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, α-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed. Received: January 22, 1998 / Accepted: February 16, 1998     

Proton-coupled bioenergetic processes in extremely alkaliphilic bacteria

Oxidative phosphorylation, which involves an exclusively proton-coupled ATP synthase, and pH homeostasis, which depends upon electrogenic antiport of cytoplasmic Na⁺ in exchange for H⁺, are the two known bioenergetic processes that require inward proton translocation in extremely alkaliphilic bacteria. Energy coupling to oxidative phosphorylation is particularly difficult to fit to a strictly chemiosmotic model because of the low bulk electrochemical proton gradient that follows from the maintenance of a cytoplasmic pH just above 8 during growth at pH 10.5 and higher. A large quantitative and variable discrepancy between the putative chemiosmotic driving force and the phosphorylation potential results. This is compounded by a nonequivalence between respiration-dependent bulk gradients and artificially imposed ones in energizing ATP synthesis, and by an apparent requirement for specific respiratory chain complexes that do not relate solely to their role in generation of bulk gradients. Special features of the synthase may contribute to the mode of energization, just as novel features of the Na⁺ cycle may relate to the extraordinary capacity of the extreme alkaliphiles to achieve pH homeostasis during growth at, or sudden shifts to, an external pH of 10.5 and above.     

Alkaliphiles — from an industrial point of view

Abstract: Organisms with pH optima for growth in excess of pH 9 are defined as alkaliphiles (or sometimes alkalophiles). Alkaliphiles contain prokaryotes, eukaryotes, and archaea. It is clear that many different taxa are represented among the alkaliphiles, and some of them are proposed as new taxa. Alkaliphiles can b isolated from normal environments such as garden soil, although counts of the alkaliphiles are higher in alkaline environments. Alkaliphiles have made a great impact in industrial applications. Biological detergents contain alkaline enzymes, such as alkaline cellulases and/or alkaline proteases that have been produced from alkaliphiles. The current proportion of total world enzyme production destined for the laundry detergents market exceeds 30%. Another important application is the industrial production of cyclodextrin with alkaline cyclomaltodextrin glucanotransferase. This enzyme reduced the production cost and paved the way for its use in large quantities in foodstuffs, chemicals and pharmaceuticals. Besides these applications, there are other possible applications in food and waste treatment industries.     

A catalytic carbohydrate contributes to bacterial antibiotic resistance

Penicillins are widespread in nature and lethal to growing bacteria. Because of the severe threat posed by these antibiotics, bacteria have evolved a wide variety of strategies for combating them. Here, we describe one unusual strategy that involves the activity of a catalytic carbohydrate. We show that the cyclic oligosaccharide, β-cyclodextrin (βCD), can hydrolyze, and thereby inactivate, penicillin in vivo. Moreover, we demonstrate that this catalytic activity contributes to the antibiotic resistance of a bacterium that synthesizes this oligosaccharide in the laboratory. Taken together, these data not only expand our understanding of the biochemistry of penicillin resistance, but also provide the first demonstration of natural carbohydrate-mediated catalysis in a living system. Paul de Figueiredo, Becky Terra and Jasbir Kaur Anand have contributed equally to this work.     

Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.