Genome sequence analysis and bioactivity profiling of marine-derived actinobacteria, Brevibacterium luteolum, and Cellulosimicrobium funkei.

Genome analysis gives important insights into the biosynthetic potential of marine actinobacteria. The genomes of two marine actinomycetes Brevibacterium luteolum MOSEL-ME10a and Cellulosimicrobium funkei MOSEL-ME6 were sequenced to identify the biosynthetic gene clusters (BGCs). Additionally, anti-proliferative, antioxidant, and enzyme inhibitory activities were studied in vitro. We report a total genome size of 2.77 Mb with GC content of 67.8% and 6.81 Mb with GC content of 69% for Brevibacterium sp. MOSEL-ME10a and Cellulosimicrobium sp. MOSEL-ME6, respectively. Biosynthetic gene clusters (BGCs) encoding different classes of natural products were predicted including terpenes, peptides, siderophores, ectoines, and bacteriocins. The bioactivity potential of crude extracts derived from these strains was evaluated. Notable anti-proliferative activity was observed against HepG2 cell line (hepatocellular carcinoma) with an IC50 value of 182 µg/mL for Brevibacterium sp. MOSEL-ME10a. Furthermore, antioxidant activity was assessed with IC50 values of 48.91 µg/mL and 102.5 µg/mL for Brevibacterium sp. MOSEL-ME10a and Cellulosimicrobium sp. MOSEL-ME6, respectively. Protein kinase inhibition potential was observed only for Brevibacterium sp. MOSEL-ME10a. Our study also reports lower amylase enzyme inhibition potential for both strains. Moreover, both crude extracts showed only slight-to-no toxic effect on erythrocytes at 400 µg/mL and below, indicating erythrocyte membrane stability. Our data present the genomic features revealing biosynthetic potential of marine actinobacteria as well as biological activities found in vitro.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

Plant growth promoting bacteria from Crocus sativus rhizosphere.

Present study deals with the isolation of rhizobacteria and selection of plant growth promoting bacteria from Crocus sativus (Saffron) rhizosphere during its flowering period (October-November). Bacterial load was compared between rhizosphere and bulk soil by counting CFU/gm of roots and soil respectively, and was found to be ~40 times more in rhizosphere. In total 100 bacterial isolates were selected randomly from rhizosphere and bulk soil (50 each) and screened for in-vitro and in vivo plant growth promoting properties. The randomly isolated bacteria were identified by microscopy, biochemical tests and sequence homology of V1-V3 region of 16S rRNA gene. Polyphasic identification categorized Saffron rhizobacteria and bulk soil bacteria into sixteen different bacterial species with Bacillus aryabhattai (WRF5-rhizosphere; WBF3, WBF4A and WBF4B-bulk soil) common to both rhizosphere as well as bulk soil. Pseudomonas sp. in rhizosphere and Bacillus and Brevibacterium sp. in the bulk soil were the predominant genera respectively. The isolated rhizobacteria were screened for plant growth promotion activity like phosphate solubilization, siderophore and indole acetic acid production. 50 % produced siderophore and 33 % were able to solubilize phosphate whereas all the rhizobacterial isolates produced indole acetic acid. The six potential PGPR showing in vitro activities were used in pot trial to check their efficacy in vivo. These bacteria consortia demonstrated in vivo PGP activity and can be used as PGPR in Saffron as biofertilizers.This is the first report on the isolation of rhizobacteria from the Saffron rhizosphere, screening for plant growth promoting bacteria and their effect on the growth of Saffron plant.

Brevibacterium daeguense sp. nov., a nitrate-reducing bacterium isolated from a 4-chlorophenol enrichment culture.

A Gram-reaction-positive, non-spore-forming, aerobic actinobacterial strain (2C6-41(T)) was isolated from the activated sludge from an industrial wastewater treatment plant in Daegu, South Korea. Its taxonomic position was investigated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, closest phylogenetic relatives to strain 2C6-41(T) were Brevibacterium pityocampae DSM 21720(T) (97.2 %), Brevibacterium salitolerans KCTC 19616(T) (96.7 %), Brevibacterium album KCTC 19173(T) (96.2 %) and Brevibacterium samyangense KCCM 42316(T) (96.2 %). The DNA G+C content of strain 2C6-41(T) was 66.4 mol%. Chemotaxonomic data, which included MK-8(H(2)) as the major menaquinone; meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids; ribose, mannose and glucose as major cell-wall sugars; and anteiso-C(15 : 0), anteiso-C(17 : 0), C(16 : 0) and iso-C(15 : 0) as major fatty acids, supported the affiliation of strain 2C6-41(T) to the genus Brevibacterium. The aromatic ring cleavage enzyme catechol 1,2-dioxygenase was not detected in strain 2C6-41(T), but catechol 2,3-dioxygenase was detected. The results of physiological and biochemical tests, and the low level of DNA-DNA relatedness to the closest phylogenetic relative enabled strain 2C6-41(T) to be differentiated genotypically and phenotypically from recognized species of the genus Brevibacterium. The isolate is therefore considered to represent a novel species in the genus Brevibacterium, for which the name Brevibacterium daeguense sp. nov. is proposed. The type strain is 2C6-41(T) (=KCTC 19800(T) = JCM 17458(T)).

Growth of aerobic ripening bacteria at the cheese surface is limited by the availability of iron.

The microflora on the surface of smear-ripened cheeses is composed of various species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. The objective of the present study was to show that iron availability is a limiting factor in the growth of typical aerobic ripening bacteria in cheese. For that purpose, we investigated the effect of iron or siderophore addition in model cheeses that were coinoculated with a yeast and a ripening bacterium. Both iron and the siderophore desferrioxamine B stimulated the growth of ripening bacteria belonging to the genera Arthrobacter, Corynebacterium, and Brevibacterium. The extent of stimulation was strain dependent, and generally, the effect of desferrioxamine B was greater than that of iron. Measurements of the expression of genes related to the metabolism of iron by Arthrobacter arilaitensis Re117 by real-time reverse transcription-PCR showed that these genes were transcribed during growth in cheese. The addition of desferrioxamine B increased the expression of two genes encoding iron-siderophore ABC transport binding proteins. The addition of iron decreased the expression of siderophore biosynthesis genes and of part of the genes encoding iron-siderophore ABC transport components. It was concluded that iron availability is a limiting factor in the growth of typical cheese surface bacteria. The selection of strains with efficient iron acquisition systems may be useful for the development of defined-strain surface cultures. Furthermore, the importance of iron metabolism in the microbial ecology of cheeses should be investigated since it may result in positive or negative microbial interactions.

Global regulation of the response to sulfur availability in the cheese-related bacterium Brevibacterium aurantiacum.

In this study, we combined metabolic reconstruction, growth assays, and metabolome and transcriptome analyses to obtain a global view of the sulfur metabolic network and of the response to sulfur availability in Brevibacterium aurantiacum. In agreement with the growth of B. aurantiacum in the presence of sulfate and cystine, the metabolic reconstruction showed the presence of a sulfate assimilation pathway, thiolation pathways that produce cysteine (cysE and cysK) or homocysteine (metX and metY) from sulfide, at least one gene of the transsulfuration pathway (aecD), and genes encoding three MetE-type methionine synthases. We also compared the expression profiles of B. aurantiacum ATCC 9175 during sulfur starvation or in the presence of sulfate. Under sulfur starvation, 690 genes, including 21 genes involved in sulfur metabolism and 29 genes encoding amino acids and peptide transporters, were differentially expressed. We also investigated changes in pools of sulfur-containing metabolites and in expression profiles after growth in the presence of sulfate, cystine, or methionine plus cystine. The expression of genes involved in sulfate assimilation and cysteine synthesis was repressed in the presence of cystine, whereas the expression of metX, metY, metE1, metE2, and BL613, encoding a probable cystathionine-γ-synthase, decreased in the presence of methionine. We identified three ABC transporters: two operons encoding transporters were transcribed more strongly during cysteine limitation, and one was transcribed more strongly during methionine depletion. Finally, the expression of genes encoding a methionine γ-lyase (BL929) and a methionine transporter (metPS) was induced in the presence of methionine in conjunction with a significant increase in volatile sulfur compound production.

Purification and gene cloning of an enantioselective thioesterification enzyme from Brevibacterium ketoglutamicum KU1073, a deracemization bacterium of 2-(4-chlorophenoxy)propanoic acid.

We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg(2+), MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the K(m) and k(cat) values for (S)-CPPA were 0.92 ± 0.17 mM and 0.28 ± 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C(8)). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.

[Biosynthesis of L-lysine-alpha-oxidase, an antitumor enzyme, by Trichoderma spp].

Strains of Trichoderma spp., producing L-lysine-alpha-oxidase, prospective in chemotherapy of malignant tumors, were studied. The best results of the enzyme biosynthesis were observed on the wheat bran medium. When grown on the media with various pH levels, the strains showed different spectra of the L-aminooxidase activity. The highest activity of the strains was recorded with respect to destruction of L-lysine. The lysine-producing organism Brevibacterium sp. induced L-lysine-alpha-oxidase activity in Trichoderma spp.

The effect of cysteine on production of volatile sulphur compounds by cheese-ripening bacteria.

The effect of cysteine on the ability of smear cheese-ripening bacteria (Brevibacterium linens and Arthrobacter spp) to produce volatile sulphur compounds (VSC) from methionine was studied. These bacteria were cultivated in a synthetic medium supplemented with various cysteine concentrations with or without methionine. Cultures with only cysteine showed slightly lower levels of VSC produced and an unpleasant odour like rotten eggs, resulting from hydrogen sulphide production. The levels and profiles of VSC produced with supplemented methionine-cysteine mixtures had strain-dependant behaviours. However, the highest levels of dimethyl disulfide, dimethyl trisulfide and dimethyl tetrasulfide were observed when increasing the cysteine concentration from 0.2 to 1.0 gl(-1) at the same methionine concentration (1.0 gl(-1)). In contrast, production levels of thioesters, especially S-methylthio acetate, were reduced by 50 and 80% under such conditions. An initial sensory approach showed that such an effect could have a strong impact on the global odour of ripened cheeses.

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum.

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.

A microbial consortium isolated from a crude oil sample that uses asphaltenes as a carbon and energy source.

A microbial consortium capable of mineralizing asphaltenes was obtained from the Maya crude oil. The enrichment system was built with a glass column reactor containing mineral medium supplied with asphaltenes as energy and carbon source. The consortium growth was evaluated in Casoy agar during 40 weeks. The steady-state phase of the enriched bacterial community was observed after 10 weeks when the culture reach 10(5) to 10(6) CFU ml(-1). The isolates belong to bacterial genus reported for degradation of other hydrocarbons and they were identified as Corynebacterium sp., Bacillus sp., Brevibacillus sp. and Staphylococcus sp. The bacterial consortium growth was evaluated by a viable counts during 14 days exposed to different aeration, temperature, salinity, and pH conditions. The ability of the consortium to mineralize asphaltenes was evaluated using the method of ISO 9439 in glass column reactors of 20 x 3.2 cm during 13 days. Temperatures of 55 degrees C and salinity of 1.8% were growth limiting. The respiration of the microbial consortium using asphaltenes as a sole carbon source (800 micromoles CO2 in 13 days) was significantly higher than those of the samples containing only the microbial consortium (200 micromoles CO2) or only asphaltenes (300 micromoles CO2). These results indicated the existence of asphaltenes-degradating microbes in the crude oil and confirmed that the consortium could mineralize asphaltenes in conditions of room temperature, salinity of 100 ppm, aeration of 1 l min(-1) and pH of 7.4.

Characterization of an antibacterial peptide produced by Brevibacterium linens.

AIMS: The aim of this research was to investigate the antimicrobial activity produced by Brevibacterium linens ATCC 9175. METHODS AND RESULTS: A bacteriocin produced by the red smear cheese bacterium B. linens ATCC 9175 was identified. The antimicrobial activity was first produced at the exponential growth phase. A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains. It inhibited the growth of Listeria monocytogenes ATCC 7644, B. linens ATCC 9172 and Corynebacterium fimi NCTC 7547, but was inactive against the Gram-negative bacteria and yeast tested. The bacteriocin was stable at 30 degrees C but the activity was lost when the temperature reached 50 degrees C. It was sensitive to the proteolytic action of trypsin, papain and pronase E and was active between pH 6.0 and 9.0. The bacteriocin was bactericidal to L. monocytogenes at 40 AU ml(-1). Bacteriostasis was observed for a low dose of bacteriocin (20 AU ml(-1)). CONCLUSIONS: An antibacterial peptide produced by B. linens was characterized, presenting potential for use as a biopreservative in food systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a novel bacteriocin active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage micro-organisms.

The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers.

BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells. These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity. AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311. METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays. RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner. The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18. Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide. CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.

Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity.

The growth of a halotolerant Brevibacterium sp. JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives. The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity. Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl. The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer. The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl). The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.

Salvage pathway for NAD biosynthesis in Brevibacterium ammoniagenes: regulatory properties of triphosphate-dependent nicotinate phosphoribosyltransferase.

As the rate-limiting enzyme, catalyzing the first reaction in NAD salvage synthesis, nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) is of important interest for studies of intracellular pyridine nucleotide pool regulation. We have purified NAPRTase 520-fold from Brevibacterium ammoniagenes ATCC 6872 without using an over-expression system by applying acid treatment, salt fractionation, Ca-phosphate gel treatment, anion exchange column chromatography and size-exclusion gel filtration. Unlike this enzyme from other sources, B. ammoniagenes NAPRTase was found to be controlled by the feedback inhibition by the end product NAD with K(i)=0.7+/-0.1 mM. The reaction products, pyrophosphate and nicotinate mononucleotide, also decreased the enzyme activity, as did other intermediates of NAD synthesis, such as AMP, ADP and a NAD direct precursor, nicotinate adenine dinucleotide or deamido NAD. The enzyme was observed to require a nucleoside triphosphate for its activity and showed the maximum affinity for ATP. The specificity, however, turned out to be poor, and ATP could be substituted by other nucleoside triphosphates as well as by sodium triphosphate. The kinetic characteristics of the enzyme are reported. For the first time, our data have experimentally revealed such complicated stimulatory and inhibitory effects by the intermediates of NAD biosynthesis on one of its salvage enzymes, NAPRTase. On the basis of these data, the key role of NAPRTase is discussed in light of the regulation of NAD metabolism in B. ammoniagenes.

Fermentation and recovery of L-glutamic acid from cassava starch hydrolysate by ion-exchange resin column

Investigators were carried out with the aim of producing L-glutamic acid from Brevibacterium sp. by utilizing a locally available starchy substrate, cassava (Manihot esculenta Crantz). Initial studies were carried out in skate flasks, which showed that even though the yield was high with 85-90 DE (Dextrose Equivalent value), the maximum conversion yield (-34 per cent) was obtained by using only partially digested starch hydrolysate, i.e. 45-50 DE. Fermentations were carried out in batch mode in a 5 L fermenter, using suitably diluted cassava starch hydrolysate, using a 85-90 DE value hydrolysate. Media supplemented with nutrients resulted in an accumulation of 21 g/L glutamic acid with a fairly high (66,3 per cent) conversation yield of glucose to glutamic acid (based on glucose consumed and on 81,74 per cent theoretical conversion rate). The bioreactor conditions most conductive for maximum production were pH 7.5, temperature 30§C and an agitation of 180 rpm. When fermentation was conducted in fed-batch mode by keeping the residual reducing sugar concentration at 5(per cent) w/v, 25,0 g/L of glutamate was obtained after 40 h fermentation (16 per cent more the batch mode). Chromatographic separation by ion-exchange resin was used for the recovery and purification of glutamic acid. It was further crystallized and separated by making use of its low solubility at the isoelectric point (pH 3.2).

A gene (tmpA) for an efflux protein of the transporter family III from Brevibacterium linens OC2, an antibacterial substance-producing strain.

A gene (tmpA) encoding a putative transmembrane protein has been cloned from B. linens OC2, an antibacterial substance-producing strain. The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics. Northern blot analysis indicated that tmpA gene is expressed. Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon. The tmpA gene is unusual among B. linens species since it was not detected among eight B. linens collection strains and 40 B. linens industrial strains.

Carnitine acts as a compatible solute in Brevibacterium linens.

Carnitine is a trimethyl amino acid found at relatively high concentrations in materials of animal origin. Exogenously provided L-carnitine was found to stimulate growth of Brevibacterium linens ATCC 19391 in media with inhibitory osmotic strength. Its osmoprotective ability was as potent as that of glycine betaine. Electrophoretic and spectroscopic (NMR) analysis showed that this compound is only transiently accumulated, but in significant amounts, by B. linens under hyperosmotic stress and is converted into glycine betaine. The L-carnitine/glycine betaine pathway is inducible by L-carnitine in B. linens. The D-enantiomer did not improve growth of B. linens, even though this solute is accumulated by B. linens at the same level as glycine betaine. The two isomeric forms of carnitine repress the build-up of ectoine, the main endogenous osmolyte in B. linens.

Mode of action of linenscin OC2 against Listeria innocua.

Linenscin OC2 is a small hydrophobic substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. Linenscin OC2 inhibits growth of gram-negative bacteria with an altered outer membrane permeability and gram-positive bacteria. It is also able to lyse eucaryotic cells. The mode of action of linenscin OC2 on the Listeria innocua cytoplasmic membrane and the effects of environmental parameters were investigated. Addition of low doses of linenscin OC2 resulted in an immediate perturbation of the permeability properties of the cytoplasmic membrane and of the bacterial energetic state. Linenscin OC2 induced a loss of cytoplasmic potassium, depolarization of the cytoplasmic membrane, complete hydrolysis of internal ATP, efflux of inorganic phosphate, and transient increase in oxygen consumption. Potassium loss occurred in the absence of a proton motive force and was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains of the cytoplasmic membrane. We propose that linenscin OC2 interacts with the cytoplasmic membrane and that the permeability changes observed at low doses reflect the formation of pore-like structures in this membrane.

Molecular cloning and transcriptional analysis of a guanosine kinase gene of Brevibacterium acetylicum ATCC 953.

The Brevibacterium acetylicum gsk gene, which encodes guanosine kinase (ATP:guanosine 5'-phosphotransferase), a kinase that is involved in guanosine salvage pathways, has been cloned by using the N-terminal amino acid sequence of the purified protein. The cloned chromosomal fragment containing the gsk gene was sequenced and shown to encode a polypeptide of 303 amino acids with a molecular mass of 32,536 Da, which is in good agreement with the measured molecular weight of the purified enzyme. Recombinant Escherichia coli strains harboring plasmids carrying the B. acetylicum gsk gene overexpressed both guanosine and inosine kinase activities. The primary structure of the gsk gene shows similarity to amino acid sequences of sugar kinases classified in the ribokinase family stronger than to those of the E. coli gsk gene encoding guanosine kinase and other nucleoside kinases. Northern blot analysis and primer extension analysis revealed a 1.4-kb transcript and promoter sequences, like the E. coli sigma70 and B. subtilis sigmaA consensus sequences, respectively. These results, together with the nucleotide sequence of the downstream region of gsk, suggested that the organization of B. acetylicum gsk is bicistronic. The second gene, orf2, shows significant similarity to the mutT mutator genes of several organisms, although its function has not yet been identified. The gsk gene was specifically transcribed in the early exponential growth phase, which seems to correspond to the specific guanosine kinase activity profile and suggests a role in controlling the nucleoside monophosphate level by efficiently recycling guanosine when cells are in the early exponential phase.