Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase     

Interspecific plasmid transfer between Streptococcus pneumoniae and Bacillus subtilis

Summary The streptococcal plasmids pMV158 and pLS1, grown in Streptococcus pneumoniae, were transferred to Bacillus subtilis by DNA-mediated transformation. The plasmids were unchanged in the new host; no deletions were observed in 80 instances of transfer. Their copy number was similar to that in S. pneumoniae. Two B. subtilis plasmids, pUB110 and pBD6, could not be transferred to S. pneumoniae. Hybrid plasmids were produced by recombining the EcoRI fragment of pBD6 that confers Km^r with EcoRI-cut pLS1, which confers Tc^r. The simple hybrid, pMP2, was transferable to both species and expressed Tc^r and Km^r in both. A derivative, pMP5, which contained an insertion in the pBD6 component, expressed a higher level of kanomycin resistance and was more easily selected in S. pneumoniae. Another derivative, pMP3, which contained an additional EcoRI fragment, presumably of pneumococcal chromosomal DNA, could not be transferred to B. subtilis. Previous findings that monomeric plasmid forms could transform S. pneumoniae but not B. subtilis were confirmed using single plasmid preparations. Although plasmids extracted from either species were readily transferred to S. pneumoniae, successive passage in B. subtilis increased the ability of plasmid extracts to transfer the plasmid to a B. subtilis recipient. This adaptation was tentatively ascribed to an enrichment of multimeric forms in extracts of B. subtilis as compared to S. pneumoniae. A review of host ranges exhibited by plasmids of Gram-positive bacteria suggested differences in their ability to use particular host replication functions. The pMP5 plasmid, with readily selectable Km^r and Tc^r markers in both hosts, and with the potential for inactivation of Km^r by insertion in the Bg/II site, could be a useful shuttle vector for cloning in S. pneumoniae and B. subtilis.     

Construction of Broad Host Range Cloning Vectors for Gram-negative Bacteria

A new cloning vector, pMFY31, has been constructed based on the high-copy-number, broad-host-range plasmid RSF1010. The plasmid has a size of 13.2 kb and carries the Ap^r, Cm^r, and Tc^r genes. It contains unique PstI, EcoRI, HindIII, BamHI, and SalI sites, all of which are located within the antibiotic resistance genes, therefore all sites are applicable to insertional inactivation. We also constructed pMFY40, a 11.6 kb derivative of pMFY31, by the elimination of the Cm^r gene. Plasmid pMFY31 has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer with the helper plasmid, and was maintained stably in the strain.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Distribution of Tetracycline Resistance Genes and Transposons among Phylloplane Bacteria in Michigan Apple Orchards

The extent and nature of tetracycline resistance in bacterial populations of two apple orchards with no or a limited history of oxytetracycline usage were assessed. Tetracycline-resistant (Tc^r) bacteria were mostly gram negative and represented from 0 to 47% of the total bacterial population on blossoms and leaves (versus 26 to 84% for streptomycin-resistant bacteria). A total of 87 isolates were screened for the presence of specific Tc^r determinants. Tc^r was determined to be due to the presence of Tet B in Pantoea agglomerans and other members of the family Enterobacteriacae and Tet A, Tet C, or Tet G in most Pseudomonas isolates. The cause of Tc^r was not identified in 16% of the isolates studied. The Tc^r genes were almost always found on large plasmids which also carried the streptomycin resistance transposon Tn5393. Transposable elements with Tc^r determinants were detected by entrapment following introduction into Escherichia coli. Tet B was found within Tn10. Two of eighteen Tet B-containing isolates had an insertion sequence within Tn10; one had IS911 located within IS10-R and one had Tn1000 located upstream of Tet B. Tet A was found within a novel variant of Tn1721, named Tn1720, which lacks the left-end orfI of Tn1721. Tet C was located within a 19-kb transposon, Tn1404, with transposition genes similar to those of Tn501, streptomycin (aadA2) and sulfonamide (sulI) resistance genes within an integron, Tet C flanked by direct repeats of IS26, and four open reading frames, one of which may encode a sulfate permease. Two variants of Tet G with 92% sequence identity were detected.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Mercury-resistant plasmids in bacteria from a mercury and antimony deposit area

Summary Most bacterial cells (Pseudomonas, Acinetobacter) obtained from the soil at the Khaidarkan mercury and antimony mine (Kirghiz USSR) contain R plasmids with mercury (HgCl₂) resistance determinants. The plasmids have a large molecular mass (about 100 MD, though smaller ones also occur), and at least some of them are transmissive. Many of the Hg^r bacteria also display an elevated antimony (SbCl₃) resistance, though this trait was not shown to be plasmid-dependent. There are practically no Hg^r plasmids in bacteria taken from the soil at different distances from the mine: the saturation of bacteria with Hg^r plasmids is maintained by selective pressure only in the area with a high enough toxin concentration.In the same mercury and antimony deposit area Hg^r plasmids were also found in Escherichia coli isolates from the gut of Mus musculus mice and Bufo viridis toads. At least some of the bacterial plasmids obtained from animals also carry antibiotic-resistance determinants (Tc^r, Cm^r, Sm^r). These plasmids are also transmissive. They display internal instability and lose their resistance determinants after a conjugation transfer to other E. coli trains.     

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in Pneumococcus

Genes for chloramphenicol resistance (Cm^r) and tetracycline resistance (Tc^r), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cm^r and Tc^r was the most common event. Tetracycline resistance was transferred alone from one Tc^r strain or rarely from Cm^rTc^r donors, whereas Cm^r was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cm^r or Tc^r determinants. No transfer of Cm^r or Tc^r toStreptococcus faecalis JH2-2 was observed.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Characterization and transferability of Clostridium perfringens plasmids.

Two strains of Clostridium perfringens resistant to clindamycin (Cl), chloramphenicol (Cm), erythromycin (Em), and tetracycline (Tc) were isolated in France in 1974 and 1975. For one of these strains, curing experiments and molecular characterization of the extrachromosomal DNA clearly demonstrate the existence of two plasmids, plP401 (54 kilobases) and plP402 (63 kilobases), which, respectively, code for Tc Cm and Em Cl resistance. With mixed cultures, the Tc Cm plasmid is transferable to sensitive strains of C. perfringens; a segregation of these markers is frequently observed during mating experiments. In contrast, the transfer of the naturally occurring plasmid Em Cl does not occur at a significant rate. In performing transfer experiments in axenic mice, we obtained a Cl^r Em^r Tc^r transcipient whose chromosomal properties are those of a hybrid. When used in mating as a parental strain, this strain promotes chromosomal gene exchange. The role of the plasmid in this phenomenon is discussed, these transcipients being generally Cl^r Em^r Tc^r. The plasmid transfer is not limited to antibiotic resistance plasmids, the transferability of a bacteriocinogenic plasmid, plP404, harbored by C. perfringens BP6K-N5 being shown also. The transfer mechanism remains to be proved; it might be a conjugation process, a cell-to-cell contact being necessary for the transfer.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)     

Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Summary The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cm^r structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P₁, P₂, P₃, P_a, P_b, P_hs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P₁ can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.Abbreviations Ap^r ampicillin resistance - CAT chloramphenicol acetyl transferase - Cm^r chloramphenicol resistance - kb kilobase pair - orf open reading frame - P promoter - T terminator - Tc^r tetracycline resistance     

Mercury resistance determined by a self-transmissible plasmid inBacillus cereus 5

Summary Inducible mercuric reductase activity inBacillus cereus 5 was plasmid-encoded. Plasmid analysis revealed three plasmids with molecular masses of 2.6, 5.2 and 130 MDa. A mating system permitted transfer of the resistance determinant among strains ofB. cereus andB. thuringiensis. Transfer of mercury resistance fromB. cereus 5 toB. cereus 569 andB. thuringiensis occurred during mixed culture incubation on agar surfaces. The 130-MDa plasmid (pGB130) was responsible for transfer; frequencies ranged from 10^–5 to 10^–4.B. cereus 569 transconjugants inheriting pGB130 were also effective donors. High transfer frequencies and the finding that cell-free filtrates of donor cultures were ineffective in mediating transfer suggested mercury-resistance transfer was not phage-mediated. Transfer was also insensitive to DNase activity. Further evidence that pGB130 DNA carried the mercury-resistance determinant was transformation ofB. cereus 569 by electroporation with pGB130 DNA isolated fromB. cereus 5 and a mercury-resistantB. cereus 569 transconjugant. Mercury-resistant transconjugants and transformants exhibited mercuric reductase activity. Plasmid pGB130 also conferred resistance to phenylmercuric acetate.     

Transformation of a new Gram-positive methylotroph,Brevibacterium methylicum,by plasmid DNA

Summary Brevibacterium methylicum is a newly isolated Gram-positive facultatively methylotrophic bacterium that uses the NAD⁺-dependent methanol dehydrogenase for methanol oxidation and assimilates its carbon via the ribulose monophosphate cycle. Protoplasts prepared by lysozyme treatment of B. methylicum cells grown in the presence of glycine were transformed by plasmid shuttle vectors pCEM500 (16.5 kb; Sm^r/Sp^r, Km^r/Gm^r) and pEC71 (7.1 kb; Km^r/Nm^r) constructed on the basis of B. lactofermentum plasmid pAM330 and replicating in Escherichia coli and in amino-acid-producing coryneform bacteria. The resistance markers were found to be expressed in B. methylicum and autonomous plasmid DNAs of various size were isolated from the transformants. The presence of the pAM330 replicon in these plasmids was demonstrated by DNA-DNA hybridization experiments. Offprint requests to: J. Nevera     

Prevalence of tetracycline resistance genes in Greek seawater habitats

The presence of selected tetracycline resistance (Tc^R) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, Tc^R bacteria, and exogenous isolated plasmids conferring Tc^R. The direct DNA-based analysis showed that tet(A) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fish-farm and coastal samples. Resistance genes tet(h), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the Tc^R genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring Tc^R genes in all the habitats tested. Plasmids were shown to carry tet(h), tet(C), tet(E), and tet(K) genes. It is concluded that Tc^R genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities.     

Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker

Incompatibility relationships between staphylococcal plasmids carrying the same, single resistance marker were studied by means of appropriate recombinant plasmids. Naturally occurring plasmids encoding streptomycin, tetracycline, or chloramphenicol resistance, respectively, were used in this study, four of each phenotype. The plasmids responsible for tetracycline resistance proved to belong to a single incompatibility set. Similarly, the four streptomycin resistance plasmids fall in the same incompatibility set. On the other hand, plasmids encoding chloramphenicol resistance were divided in four distinct incompatibility sets, three of them being newly defined. Study of the molecular relationships between these plasmids by DNA-DNA hybridization and restriction endonuclease cleavage supported the conclusions from genetic tests that the four Tc^r and the four Sm^r plasmids are essentially identical, whereas the four Cm^r plasmids are diverse.     

Plasmid maintenance and threonine production by recombinantEschcrichia coli strains

Summary E. coli NRRL 12100-a recombinant strain obtained by genetic manipulationwas used forL-threonine production. When cultured in a rich medium without antibiotics three types of colonies were isolated (Ap^sTc^s, Ap^rTc^s, and Ap^rTc^r). The Ap^rTc^r clones were best threonine producers (9 to 12 g/1) and the plasmid was maintained, in 65 to 93% of the host cells. When inocula were grown under selective pressure we obtained about 10 g/1 of threonine and a plasmid maintenance of 83%. When growth of inocula was done without antibiotic, threonine yields dropped to 5 g/1 and 64% of the cells have lost the plasmid. Batch culture experiments were performed with 3, 4 and 6% of glucose, added at the initial stage or in a discontinuous feed. Threonine yields and plasmid stability were not affected. The elimination of the maximum level of threonine produced (from 13.8 to 6.7 g/1) and on the plasmid maintenance (from 94 to 4% of the cells) while growth of the strain was not affected.     

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc^r) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc^r genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc^r genes was quantified by real-time quantitative PCR. To confirm the Tc^r gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc^r genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc^r genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.