Sporosarcina Species Research Articles

Urban biomining: lithium recovery from spent batteries through multi-step bioprocesses

Multi-step design to evaluate the bio-recovery of lithium from spent batteries was studied. The first step consisted of lithium extraction from spent batteries, using bacterial and fungal acid extrolites. The second step explored lithium recovery in the form of carbonate salts by using MICP (Microbial Induced Carbonate Precipitation) bacteria from Sporosarcina species. For lithium extraction (Step 1) sulfuric acid produced by sulphur oxidizing bacteria Acidithiobacillus thiooxidans was evaluated for its capacity to leach lithium. Extraction with biogenic sulfuric acid and with fungal bio- produ-cts (from Aspergillus sp. and Simplicillum sp. isolated at our facilities) were compared with commercial sulfuric acid. For biorecovery processes (Step 2), two type strains of Sporosarcina sp. were tested due to their capacity to precipitate lithium carbonate. Results showed fungal bioextracts gave a lithium leaching yield close to 60% and a global recovery yield of 27%. These observations are reported for the first time and lay the foundations for continuing the study and scaling up of this combined process for lithium recovery.

Biomineralization Grouting for Beach Sand Cemented with MICP

Microbial-induced carbonate precipitation (MICP) is an environmentally friendly approach that relies on the production of calcium carbonate by microorganisms to construct or reinforce coastal structures. In order to address the disadvantages of current coastal countermeasures techniques, MICP is a cost-effective solution that can be used to repair and restore coastal habitats damaged by human activities. The resulting structures formed through MICP are strong and durable, providing long-term protection against erosion and flooding caused by storms or rising sea levels. Biominerals, including calcium carbonate or calcium phosphate, are used to create complex composites with organic molecules by combining the strength of inorganic materials with the versatility and biocompatibility of organic macromolecules. It is of the utmost importance to investigatethe functionality of MICP and scale up its deployment in various fields in order to thoroughly assess the instrument’s application. Coastal erosion has been a severe concern in archipelagic countries. Therefore, this study explored the Miyazaki coast in Japan and the Yogyakarta coastline in Indonesia to minimize coastal erosion using MICP. The bacteria found in Miyazaki (Sporosarcina species) and the Yogyakarta coast (Pseudoalteromonas tetradonis) were used in the experiment. As a result, the sample treated with a gradual injection of the cementation solution achieved about 6 MPa UCS after 21 days of treatment. The objective were investigated the potential biotreatment with original sand materials and to evaluate the long-term durability under saturated conditions. For these purposes, the MICP-treated sand columns were subjected to series of compression tests and wet-drying (WD) durability analysis.

Draft genome of Sporosarcina sp. NCCP-2222T isolated from rhizosphere of mangrove plant Ceriops tagal reveals its potential as a plant biostimulant and constitutes a novel species

Reports on Sporosarcina species associated with mangroves native plants and their diversity are scarced. Draft genome of Sporosarcina sp. strain NCCP-2222T, isolated from the rhizosphere of Ceriops tagal, a mangrove plant from the Lasbela, Pakistan, is presented. The assembled genome of NCCP-2222T features genome size of 4,019,411 bp, DNA G+C content of 44.4%, and predicted 3990 protein coding genes. The digi-DDH and ANI analyses indicated that strain NCCP-2222T represents a novel species in genus Sporosarcina. Genome mining disclosed the presence of genes involved in solubilization of phosphorus and production of indole acetic acid (IAA) in strain NCCP-2222T. Draft genome of Sporosarcina sp. NCCP-2222T reveals insights on its potential as a plant biostimulant and provides evidence to represent it as a novel species, expanding our knowledge regarding Sporosarcina biodiversity worldwide and specifically those strains associated with native mangroves plants in Pakistan.

Study of As-resistant bacteria from Nadia, India and a survey of two As resistance-related proteins.

The present investigation deals with the characterisation of three As-resistant bacteria, Bacillus aryabhattai strain VPS1, Bacillus licheniformis strain VPS6 and Sporosarcina thermotolerans strain VPS7 isolated from the rhizosphere of a contaminated paddy field in Chakdaha, Nadia, West Bengal, India. Two strains, VPS6 and VPS7 showed ureolytic activity, which can be used for microbial-induced calcite precipitation of As as a bioremediation option. However, As reduction and oxidation capacities were not reported in any of these bacteria. A phylogenetic tree of 16S ribosomal RNA gene sequences was constructed for all three bacterial isolates, including different species of As-resistant Bacillus and Sporosarcina. Furthermore, literature survey and genome mining were employed to explore the diversity of As resistance-related proteins, arsenite S-adenosylmethyltransferase (ArsM) and arsenical pump membrane protein (ArsB) among different bacteria, and the phylogenetic relatedness was studied to understand the distribution and evolution of their amino acid sequences. ArsB was predominantly present in a wide variety of bacteria (347 taxa); however, ArsM was reported in comparatively fewer isolates (109 taxa). There were a total of 60 similar taxa that contained both ArsM and ArsB. Both proteins were most abundantly present in phylum Proteobacteria. Overall, this investigation enumerates As-resistant bacteria to understand the As metabolism in the environment, and the phylogenetic analysis of As resistance-related proteins helps in understanding the functional relationship in different bacteria for their role in As mobility in the environment.

Effects of alkyl gallates, fatty acids, and acylglycerols on the growth of the psychrotolerant bacterium Sporosarcina sp. S92h

Abstract Sporosarcina species are psychrotolerant endospore-forming bacteria that are predominantly isolated from processed minced fish meat (surimi). Their levels should be controlled to maintain the quality of surimi-based food products. Our previous study suggested that the fatty acid composition of the cell membrane is an important factor for the growth of Sporosarcina species under cold conditions. In this study, we examined the effects of alkyl gallates, fatty acids, and acylglycerols on the growth of Sporosarcina sp. S92h in TSB medium at 10 °C and 30 °C. The results illustrated that the alkyl gallates isoamyl gallate, octyl gallate, and lauryl gallate; the fatty acids oleic acid, linoleic acid, and α-linolenic acid; and the acylglycerol monolaurin inhibited the growth of Sporosarcina sp. S92h for 10 days at 10 °C at a concentration of 0.2 mM. We found that compounds that exerted significant growth-inhibitory effects have similar molecular weights (range, 240.3–338.4) and ACD/LogP values (range, 3.48–7.70). These data will be useful for exploring more effective compounds to inhibit the growth of Sporosarcina species and contribute to quality control for surimi-based foods.

Modulation of fatty acid composition and growth in Sporosarcina species in response to temperatures and exogenous branched-chain amino acids.

Psychrotolerant endospore-forming Sporosarcina species have been predominantly isolated from minced fish meat (surimi), which is stored under refrigeration after heat treatment. To develop a better method for preserving surimi-based food products, we studied the growth and fatty acid compositions of the isolated strain S92h as well as Sporosarcina koreensis and Sporosarcina aquimarina at cold and moderate temperatures. The growth rates of strain S92h and S. koreensis were the fastest and slowest at cold temperatures, respectively, although these strains grew at a similar rate at moderate temperatures. In all three strains, the proportions of anteiso-C15:0 and unsaturated fatty acids (UFAs) were significantly higher at cold temperatures than at moderate temperatures. Furthermore, supplementation with valine, leucine, and isoleucine resulted in proportional increases in iso-C16:0, iso-C15:0, and anteiso-C15:0, respectively, among the fatty acid compositions of these strains. The proportions of the UFAs were also altered by the supplementation. At cold temperatures, the growth rates of strain S92h and S. koreensis, but not of S. aquimarina, were affected by supplementation with leucine. Supplementation with isoleucine enhanced the growth of S. koreensis at cold temperatures but not that of the other strains. Valine did not affect the growth of any strain. These results indicate that anteiso-C15:0 and UFAs both play important roles in the cold tolerance of the genus Sporosarcina and that these bacteria modulate their fatty acid compositions in response to the growth environment.

Calcium Carbonate Precipitation by Bacillus and Sporosarcina Strains Isolated from Concrete and Analysis of the Bacterial Community of Concrete.

Microbially induced calcium carbonate precipitation (CCP) is a long-standing but re-emerging environmental engineering process for production of self-healing concrete, bioremediation, and long-term storage of CO2. CCP-capable bacteria, two Bacillus strains (JH3 and JH7) and one Sporosarcina strain (HYO08), were isolated from two samples of concrete and characterized phylogenetically. Calcium carbonate crystals precipitated by the three strains were morphologically distinct according to field emission scanning electron microscopy. Energy dispersive X-ray spectrometry mapping confirmed biomineralization via extracellular calcium carbonate production. The three strains differed in their physiological characteristics: growth at alkali pH and high NaCl concentrations, and urease activity. Sporosarcina sp. HYO08 and Bacillus sp. JH7 were more alkali- and halotolerant, respectively. Analysis of the community from the same concrete samples using barcoded pyrosequencing revealed that the relative abundance of Bacillus and Sporosarcina species was low, which indicated low culturability of other dominant bacteria. This study suggests that calcium carbonate crystals with different properties can be produced by various CCP-capable strains, and other novel isolates await discovery.

Isolation and characterization of psychrotolerant endospore-forming Sporosarcina species associated with minced fish meat (surimi)

We studied the changes of resident microbiota in surimi—minced fish meat—during heat-treatment and subsequent cold-storage via the sequencing of partial 16S rRNA gene. Raw surimi made from Alaska pollock, pike conger, and white croaker was contaminated with 104 to 106CFU/g of various non-endospore-forming bacteria. Immediately after heat-treatment, the bacterial counts were significantly reduced to less than 1CFU/g, and only endospore-forming bacteria, identified as Bacillus species were retrieved. Subsequently, the bacterial counts increased up to 10 to 105CFU/g in the heated surimi after refrigerated storage at 5°C for 2weeks or at 10°C for 1week. Most of the isolates from the refrigerated surimi were identified as Sporosarcina species. The Sporosarcina isolates have an increased ability to grow at 10°C than the isolates related to the other endospore-forming bacteria, such as Bacillus, Lysinibacillus, and Paenibacillus species. Endospores of the Sporosarcina isolates were able to germinate and proliferate in a fish-paste product model system stored at 10°C within 8days. In order to study the cold-adaptation mechanism of Sporosarcina species, the fatty acid composition of the isolates was analyzed. At the growth temperature of 10°C, the proportions of unsaturated to saturated fatty acids and anteiso to iso fatty acids were higher than those at 28°C. The alteration of the fatty acid composition suggests that Sporosarcina species adapt to cold by maintaining the fluidity of the cell membrane because unsaturated and anteiso fatty acids have lower melting points than saturated and iso fatty acids, respectively. We concluded that the endospores of Sporosarcina species are widely distributed in surimi, and that they can survive heat-treatment and proliferate during cold-storage in fish-paste products. Controlling Sporosarcina species would contribute to improving the quality of surimi product.

Bacterial and Fungal Communities in Pu'er Tea Samples of Different Ages

Pu'er is a major kind of postfermented tea and is made with a "large leaf" variety of Camellia sinensis (C. sinensis assamica), whose distribution is limited to the mountains of southern Yunnan, China. The quality of Pu'er tea is believed to increase with storage (aging, maturing) because of postfermentation by microbes. The effect of storage period (from < 1 to 192mo) on the bacteria and fungi in Pu'er tea was investigated by a culture-dependent and a PCR-DGGE method. The individual numbers of fungi and bacteria decreased with increasing storage time and were significantly greater in ripened tea than in raw Pu'er tea. Both methods indicated that yeast, Aspergillus spp., and Penicillium spp. were the dominant fungi in almost all the samples. However, the common bacteria detected by the culture-dependent method were species of Pseudomonas, Achromobacter, Alcaligenes, Sporosarcina, and Bacillus, whereas those detected by PCR-DGGE were species of Staphylococcus, Arthrobacter, and Streptomyces. According to ordination analysis, bacterial community structure differed between ripened and raw Pu'er tea. Bacterial diversity was positively correlated with aging time, while fungal diversity in both raw and ripened tea increased during the first 60 mo of aging and then decreased. Changes in polyphenol content were correlated with the changes in fungal diversity. These results suggest that the relationship between storage time and the quality of Pu'er tea is complex and involves changes in polyphenol content and microbial abundance and diversity.

Sporosarcina contaminans sp. nov. and Sporosarcina thermotolerans sp. nov., two endospore-forming species

The taxonomic positions of two Gram-positive, endospore-forming rods, strains CCUG 53915(T) and CCUG 53480(T), isolated from an industrial clean-room floor and from a human blood sample, respectively, were studied. 16S rRNA gene sequence similarity studies revealed that both isolates clearly clustered with Sporosarcina species. Strain CCUG 53915(T) was most closely related to Sporosarcina koreensis and Sporosarcina soli, showing 99.4 and 99.2 % 16S rRNA gene sequence similarities to the type strains of these species, respectively. Strain CCUG 53480(T) showed the highest 16S rRNA gene sequence similarities to the type strains of S. koreensis (98.7 %) and Sporosarcina saromensis (98.6 %). Strains CCUG 53915(T) and CCUG 53480(T) had peptidoglycan type A4alpha l-Lys-d-Glu. The quinone systems of both strains were composed predominantly of menaquinone MK-7, with small amounts of MK-8. The polar lipid profiles of both strains consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and three unidentified phospholipids. The fatty acid profiles, which comprise anteiso- and iso-branched fatty acids, supported affiliation of the two isolates to the genus Sporosarcina. The results of physiological and biochemical tests and DNA-DNA hybridization data allowed a clear phenotypic and genotypic differentiation of both strains from the most closely related Sporosarcina species. For this reason, it is proposed that strains CCUG 53915(T) (=DSM 22204(T)) and CCUG 53480(T) (=DSM 22203(T)) represent two novel species in the genus Sporosarcina, with the names Sporosarcina contaminans sp. nov. and Sporosarcina thermotolerans sp. nov., respectively.

Isolation of bacteria from the ileal mucosa of TNFdeltaARE mice and description of Enterorhabdus mucosicola gen. nov., sp. nov.

The diversity of bacteria associated with inflamed mucosa was investigated by culturing ileal samples from TNF(deltaARE) mice on a selective medium containing mucin. Among eight isolates, two strains (Mt1B3 and Mt1B8(T)) belonged to bacterial groups not yet cultured from the mouse intestine. Whereas strain Mt1B3 was identified as a member of the family Planococcaceae and is closely related to Sporosarcina species and Filibacter limicola DSM 13886(T), strain Mt1B8(T) was a novel bacterium. Based on phylogenetic analysis, strain Mt1B8(T) is a member of the family Coriobacteriaceae. The closest relatives with validly published names were Asaccharobacter celatus, Adlercreutzia equolifaciens (<96 % similarity) and Eggerthella species (<92 %). With respect to Asaccharobacter celatus and Eggerthella, the phylogenetic position of strain Mt1B8(T) was confirmed at the chemotaxonomic level by Fourier-transform infrared spectroscopic analysis. The major fatty acid of strain Mt1B8(T) is C(16 : 0) (23.9 %). Menaquinones were monomethylated. DNA-DNA relatedness between strain Mt1B8(T) and Asaccharobacter celatus DSM 18785(T) was 28 %. Strain Mt1B8(T) is a Gram-positive-staining rod that does not form spores and has a high DNA G+C content (64.2 mol%). Cells are aerotolerant but grow only under strictly anoxic conditions. They are sensitive to cefotaxime, clarithromycin, erythromycin, metronidazole, tetracycline, tobramycin and vancomycin. API and VITEK analysis showed the ability of strain Mt1B8(T) to convert a variety of amino acid derivatives. According to these findings, it is proposed to create a novel genus and species, Enterorhabdus mucosicola gen. nov., sp. nov., to accommodate strain Mt1B8(T). The type strain of Enterorhabdus mucosicola is Mt1B8(T) (=DSM 19490(T) =CCUG 54980(T)).

Description of Paenisporosarcina quisquiliarum gen. nov., sp. nov., and reclassification of Sporosarcina macmurdoensis Reddy et al. 2003 as Paenisporosarcina macmurdoensis comb. nov.

In the course of a study of the prokaryotic diversity of a landfill site in Chandigarh, India, a strain designated SK 55(T) was isolated and characterized using a polyphasic approach. Its 16S rRNA gene sequence showed closest similarity (98.3 %) to that of Sporosarcina macmurdoensis CMS 21w(T). The sequence similarity to strains of other hitherto described species of Sporosarcina was less than 95.5 %. Strain SK 55(T) contains peptidoglycan of the A4alpha type (l-Lys-d-Asp), MK-8 and MK-7 as the major menaquinones and iso-C(15 : 0) as the major fatty acid. Strain SK 55(T), Sporosarcina macmurdoensis and Sporosarcina ureae, the type species of the genus, had some polar lipids in common (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a phospholipid and an unknown lipid). However, an aminolipid, an aminophospholipid and an unknown lipid found in the former two organisms are similar, though not identical, but quite different from the profile of S. ureae. The genomic DNA G+C contents of strain SK 55(T) (46.0 mol%) and S. macmurdoensis CMS 21w(T) (44.0 mol%) are higher than those reported for the majority of species of Sporosarcina (36-42 mol%). As revealed by 16S rRNA gene sequence analysis, strain SK 55(T) and S. macmurdoensis CMS 21w(T) form a clade which is distinct from the clade occupied by other species of Sporosarcina. On the basis of phenotypic characteristics including chemotaxonomic data and analysis of the 16S rRNA gene sequence, we conclude that strain SK 55(T) should be considered as a member of a novel genus and species, for which the name Paenisporosarcina quisquiliarum gen. nov., sp. nov. is proposed. The type strain of Paenisporosarcina quisquiliarum is SK 55(T) (=MTCC7604(T) =JCM 14041(T)). S. macmurdoensis CMS 21w(T) shows more similarity in its 16S rRNA gene sequence (98.3 %), DNA G+C content and polar lipid profile to strain SK 55(T) than to S. ureae DSM 2281(T). Phylogenetically, it forms a coherent cluster with strain SK 55(T) which is separate from the Sporosarcina cluster. Moreover, iso-C(15 : 0), anteiso-C(15 : 0) and C(16 : 1)omega7c alcohol are the three major fatty acids in both S. macmurdoensis CMS 21w(T) and SK 55(T). All these data suggest that S. macmurdoensis should be a member of the genus Paenisporosarcina. However, S. macmurdoensis can be differentiated from SK 55(T) in several physiological and biochemical characteristics, especially in the patterns of oxidation and acid production from carbohydrates. The genomic relatedness of S. macmurdoensis CMS 21w(T) and strain SK 55(T) was also very low (18.0 %). It is therefore logical to transfer Sporosarcina macmurdoensis to the newly created genus as Paenisporosarcina macmurdoensis comb. nov. The type strain is CMS 21w(T) (=MTCC4670(T) =DSM 15428(T)).

Sporosarcina luteola sp. nov. isolated from soy sauce production equipment in Japan

A Gram-variable, spore-forming, motile rod, designated strain Y1T, was isolated from the hopper surface of equipment used for soy sauce production. Phylogenetic analysis based on 16S rRNA gene sequence revealed that Y1T is affiliated phylogenetically to the genus Sporosarcina, and the strain showed sequence similarities of 95.8-99.2% to those of Sporosarcina species with validly published names. The values of DNA-DNA relatedness between strain Y1T and related type strains of the genus Sporosarcina were below 27%. The major cellular fatty acids were iso-C(15:0) and anteiso-C(15:0). The cell-wall peptidoglycan was of the A4alpha type (Lys-Glu) and the major isoprenoid quinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G+C content of the strain was 43.6 mol%. On the basis of phylogenetic analysis and physiological and chemotaxonomic data, the isolate represents a novel species of the genus Sporosarcina, for which the name Sporosarcina luteola sp. nov. is proposed. The type strain is strain Y1T (= JCM 15791T = NRRL B-59180T = NBRC 105378T = CIP 109917T = NCIMB 14541T).

Sporosarcina saromensis sp. nov., an aerobic endospore-forming bacterium

Two Gram-positive, endospore-forming, rod-shaped bacterial strains, HG645(T) and HG711, were respectively isolated from surface water of a brackish lake and sediment of a fishery harbour in Japan and were subsequently characterized taxonomically using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains HG645(T) and HG711 are affiliated phylogenetically to the genus Sporosarcina, and they exhibit sequence similarities of 95.7-97.3 % to the type strains of Sporosarcina species. DNA-DNA relatedness between strain HG645(T) and the type strain of the phylogenetically related species Sporosarcina aquimarina was less than 10 %. The DNA G+C content of strains HG645(T) and HG711 were respectively 46.0 and 45.2 mol%. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The cell-wall peptidoglycan type (Lys-Glu), major cellular fatty acids (iso-C(15 : 0) and anteiso-C(15 : 0)) and quinone type (MK-7) of the isolates support their affiliation to the genus Sporosarcina. On the basis of phylogenetic analysis and physiological and chemotaxonomic data, the isolates represent a novel species of the genus Sporosarcina, for which the name Sporosarcina saromensis sp. nov. is proposed. The type strain is strain HG645(T) (=MBIC08270(T)=IAM 15429(T) =KCTC 13119(T)).

Sporosarcina koreensis sp. nov. and Sporosarcina soli sp. nov., isolated from soil in Korea

Two Gram-positive, aerobic, spore-forming rods, F73(T) and I80(T), were isolated from upland soil. A phylogenetic analysis of 16S rRNA gene sequences placed both isolates within the genus Sporosarcina, and showed a sequence similarity of 98.9 % between the two strains and a similarity of approximately 94.6-97.3 % with respect to Sporosarcina species with validly published names. The values for DNA-DNA relatedness between the two isolates and related type strains of the genus Sporosarcina were below 28.0 %. For both strains, the major cellular fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0). In both cases, the cell-wall peptidoglycan was of the A4alpha type (L-Lys-D-Glu) and the major menaquinone was MK-7. Diaminopimelic acid was absent from both strains. The genomic DNA G+C contents of strains F73(T) and I80(T) were 46.5 and 44.5 mol%, respectively. On the basis of the phylogenetic analysis and physiological and chemotaxonomic data, the isolates represent two novel species of the genus Sporosarcina, for which the names Sporosarcina koreensis sp. nov. (type strain F73(T) =KACC 11299(T) =DSM 16921(T)) and Sporosarcina soli sp. nov. (type strain I80(T) =KACC 11300(T) =DSM 16920(T)) are proposed.

Sse9I restriction-modification system: Organization of genes and structural comparison of proteins

The nucleotide sequence was established for the operon of the Sse9I type II restriction-modification system of Sporosarcina species 9D. The enzymes of the Sse9I system recognize the 5′-AATT-3′ tetranucleotide. The operon includes three genes, sse9IC-sse9IR-sse9IM, which are transcribed unidirectionally and code, respectively, for the controller protein (C.Sse9I), restriction endonuclease (R.Sse9I), and DNA methyltransferase (M.Sse9I). The region immediately upstream of sse9IC was found to contain a conserved nucleotide sequence (C box) providing a binding site for C. Sse9I. The amino acid sequences of C.Sse9I and R.Sse9I were compared with those of related proteins. In the case of R.Sse9I, the highest homology was observed with the R.MunI (5′-CAATTG-3′) and R.EcoRI (5′-GAATTC-3′) regions that harbor the amino acid residues involved in recognizing the AATT inner tetranucleotide. The sse9IR gene was cloned in an expression vector, and recombinant R.Sse 9I was isolated.

Sporosarcina macmurdoensis sp. nov., from a cyanobacterial mat sample from a pond in the McMurdo Dry Valleys, Antarctica.

Strain CMS 21w(T) was isolated from a cyanobacterial mat sample taken from a pond located in the McMurdo Dry Valleys, Antarctica. Based on its phenotypic, chemotaxonomic and phylogenetic properties, strain CMS 21w(T) was identified as a member of the genus SPOROSARCINA: At the 16S rRNA gene level, CMS 21w(T) exhibited about 93-96 % similarity to all reported species of Sporosarcina and exhibited a maximum similarity of 96 % to both Sporosarcina globispora and Sporosarcina psychrophila. Based on more than 3 % difference at the 16S rRNA gene sequence level and the presence of distinct differences with respect to phenotypic, biochemical and chemotaxonomic features, strain CMS 21w(T) (=MTCC 4670(T)=DSM 15428(T)=CIP 107784(T)) is proposed as the type strain of a novel species of Sporosarcina, Sporosarcina macmurdoensis sp. nov.

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid ( 2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO 2 using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH , reaction yields averaged 84 M%, and ee was >98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii as a source of FDH, 197 kg of 2 was produced in three batches with an average yield of 91 M% and ee > 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces PDH was used to produce 15 kg of 2 with a yield of 97 M% and >98% ee.

Cloning and characterization of Sse9I DNA-methyltransferase recognizing 5'-AATT-3'.

The gene from Sporosarcina species 9D encoding Sse9I DNA-methyltransferase (M.Sse9I) was cloned and expressed in Escherichia coli. The recombinant plasmid pMSse-1 contains the M.Sse9I gene 1086 bp in length, corresponding to a protein of 362 amino acid residues. M.Sse9I recognizes the tetranucleotide sequence 5'-AATT-3' and modifies the second adenine within the recognition sequence. The amino acid sequence of M.Sse9I was compared with those of other methylases. According to mutual positions of four conservative domains the new enzyme belongs to a subgroup of D12 class. This subgroup includes Sse9I, CviAII, NlaIII and N-terminal domains of LlaI, FokI and StsI DNA-methyltransferases.

Small, acid-soluble, spore proteins and their genes from two species of Sporosarcina

Small, acid-soluble proteins (SASP) of both the α/β- and γ-type were present in spores of Sporosarcina ureae and S. halophila, and three genes encoding α/β-type SASP in these species have been cloned and sequenced. The amino acid sequences of the Sporosarcina α/β-type SASP are extremely homologous to those of Bacillus SASP, further indicative of the close evolutionary relationship between these genera.