Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301. In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.     

Alleviation of Type I Restriction in Escherichia coli K12 Carrying the arsR Gene from pKW301 of Acidiphilium multivorum AIU 301

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is contained in pKW301. In Escherichia coli, arsR cloned under the control of P _lac in a multicopy vector alleviated restriction of nonmodified DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas in the Ard proteins the motif is in the C domain. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is –4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.     

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.     

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.     

Metal resistance in Acidocella strains and plasmid-mediated transfer of this characteristic to Acidiphilium multivorum and Escherichia coli.

Acidophilic heterotrophic strain GS19h of the genus Acidocella exhibited extremely high resistance to CdSO4 and ZnSO4, with a MIC of 1 M for each. The respective MICs for an Acidocella aminolytica strain were 400 and 600 mM. The MICs of NiSO4 for the above strains were 200 and 175 mM, respectively. These strains were also resistant to CuSO4, the MICs being 20 and 40 mM, respectively. An Acidocella facilis strain showed resistance only to ZnSO4, with a MIC of 150 mM. The metal salts, in general, extended the lag period, log period, and generation time, with decreases in growth rate and optimum growth. A. aminolytica and strain GS19h each contain more than one plasmid, while A. facilis contains none. After transformation by electroporation with the plasmid preparation from strain GS19h, an Acidiphilium multivorum strain became highly resistant to cadmium and zinc, and the plasmid profile of the transformed cells was found to differ from that of the original Acidiphilium multivorum strain. Escherichia coli HB101 and DH5 alpha also exhibited more resistance to these metals, especially zinc, after transformation with the total plasmid preparation of strain GS19h or a 24.0-MDa plasmid of the same strain, although no plasmid was detected in the transformed cells. Thus, the results derived mainly through genetic experiments demonstrate for the first time the plasmid-mediated transfer of metal resistance for an acidophilic bacterium.     

Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide.

Pseudomonas aeruginosa AI 3 was able to grow in medium containing acetanilide (N-phenylacetamide) as a carbon source when NH4+ was the nitrogen source but not when urea was the nitrogen source. AIU mutants isolated from strain AI 3 grew on either medium. Urease levels in bacteria grown in the presence of urea were 10-fold lower when NH4+ or acetanilide was also in the medium, but there were no apparent differences in urease or its synthesis between strain AI 3 and mutant AIU 1N. The first metabolic step in the acetanilide utlization is catalyzed by an amidase. Amidases in several AIU strains showed altered physiochemical properties. Urea inhibited amidase in a time-dependent reaction, but the rates of the inhibitory reaction with amidases from the AIU mutants were slower than with AI 3 amidase. The purified amidase from AIU 1N showed a marked difference in its pH/activity profile from that obtained with purified AI 3 amidase. These observations indicate that the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene; this was confirmed for strain AIU 1N by transduction.     

Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli

A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.     

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.     

Identification of non-pseudomonad bacteria from fruit bodies of wild agaricales fungi that detoxify tolaasin produced by Pseudomonas tolaasii

Bacterial isolates from wild Agaricales fungi detoxified tolaasin, the inducer of brown blotch disease of cultivated mushrooms produced by Pseudomonas tolaasii. Mycetocola tolaasinivorans and Mycetocola lacteus were associated with fruit bodies of wild Pleurotus ostreatus and wild Lepista nuda, respectively. Tolaasin-detoxifying bacteria belonging to other genera were found in various wild mushrooms. An Acinetobacter sp. was isolated from fruit bodies of Tricholoma matsutake, Bacillus pumilus was isolated from Coprinus disseminatus, and Sphingobacterium multivorum was isolated from Clitocybe clavipes. A Pedobacter sp., which seemed not be identifiable as any known bacterial species, was isolated from a Clitocybe sp. Tolaasin-detoxifying bacteria identified thus far were attached to the surface of mycelia rather than residing within the fungal cells. M. tolaasinivorans, M. lacteus, B. pumilus, the Pedobacter sp., and S. multivorum efficiently detoxified tolaasin and strongly suppressed brown blotch development in cultivated P. ostreatus and Agaricus bisporus in vitro, but the Acinetobacter sp. did so less efficiently. These bacteria may be useful for the elucidation of mechanisms involved in tolaasin-detoxification, and may become biological control agents of mushroom disease.     

Characterization of a circular plasmid from the yeast Kluyveromyces waltii.

A new plasmid was found in the yeast Kluyveromyces waltii. This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp. It has several features characteristic of the 2 mu-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms. However, the nucleotide sequence shows little homology with known yeast plasmids. An ARS function was localized within a segment of 545 bp near one of the inverted repeats. Chimeric plasmids carrying this segment efficiently transformed K. waltii. A strain of K. waltii cured of the plasmid (cir degree) was also obtained. In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability. Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir degree host and were particularly stable. pKW1-derived full-sequence plasmids also transformed K. thermotolerans, but not K. lactis.     

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.     

Nucleotide sequence analysis of cryptic plasmid pAM5 from Acidiphilium multivorum

Plasmid pAM5 of Acidiphilium multivorum JCM-8867 has been completely sequenced by initial cloning of HindIII-PstI fragments followed by primer walking. It has a size of 5161bp and single site for several restriction enzymes as revealed by DNA sequencing. Sequence analysis predicts five putative open reading frames. ORF1 and ORF3 show significant identity with various plasmid encoded mobilization (Mob) and replication initiation (Rep) proteins, respectively. The putative Mob protein has several characteristics of the MOB(Q) family having the motifs with conserved amino acid residues. Upstream of the Mob ORF, there exists a 34bp oriT region having a nic consensus sequence. The constructed plasmid pSK1 bearing pAM5 mob region can be mobilized to Escherichia coli in presence of conjugative plasmid pRK2013. The replication module comprises of several DnaA like boxes, several perfect direct and inverted repeats, a potential prokaryotic promoter and putative rep gene. The rep module is very similar to several theta replicating iteron family plasmids, suggesting pAM5 replication to follow the same course. Any phenotypic character determinant (e.g., metal resistance, antibiotic resistance etc.) gene is absent in pAM5, suggesting this plasmid to be cryptic in nature. However, a pAM5 derivative plasmid named pSK2, containing the putative pAM5 rep region, can replicate and be stably maintained in Acidiphilium, Acidocella, and E. coli strains; it can also carry foreign DNA fragments. Thus, pSK2 could serve as a cloning shuttle vector between these bacteria. It was observed that pAM5 Rep is essential for pSK2 to replicate in acidophiles. In its natural host, A. multivorum JCM-8867, pAM5 maintains a copy number of 50-60, and its derivative pSK2 maintains a comparatively, higher copy number in E. coli than in acidophiles.     

Behavior of the hybrid plasmid pNSW301 in Zymomonas mobilis grown in continuous culture

The stability of the plasmid pNSW301 which was formed by cointegration of the Inc W R plasmid Sa and the 14.5-kb pNSW1 plasmid of Zymomonas mobilis ZM6100 was investigated in ZM6100(pNSW301) grown in continuous culture without antibiotic selection. The cointegrate plasmid, pNSW301, was found to be structurally unstable and a total reduction in the size of pNSW301 of approximately 21 kb occurred during growth in continuous culture. Following a systematic study, a number of deletion derivatives of pNSW301 were isolated and used to transform Escherichia coli HB101, with the exception of pNSW312. The plasmid pNSW312 was 100% stable in ZM6100(pNSW312) in continuous culture but was unable to replicate in E. coli.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

Molecular characterization of membrane-associated soluble serine palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.     

Intramolecular transposition and inversion in plasmid R6K

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.     

Hyperproduction of tryptophan by Corynebacterium glutamicum with the modified pentose phosphate pathway.

A classically derived tryptophan-producing Corynebacterium glutamicum strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO2 yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of C. glutamicum was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers.     

Characterization of a dextranolytic biotype of Flavobacterium multivorum from soil

Bacteria obtained by enrichment on dextran as sole carbon source before plating, or by direct plating of soil suspensions on dextran agar, included a yellow, non-motile, Gram negative rod with distinctive morphology and ultrastructure in negatively stained preparations under the electron microscope. The 16 isolates obtained all showed asymmetric division. In a small proportion of the population a phenomenon resembling budding resulted in the release of spherical cells. The isolates were compared with Flavobacterium breve , in which a similar morphology has been observed, but were clearly distinct on physiological grounds. The collection of strains formed a moderately uniform phenotype similar to, but generally more active biochemically, than F. multivorum ; all produced acid from dextran and grew on dextran as sole carbon source. Seven of the isolates produced pits in pectate gel at neutral and alkaline pH, but none broke down cellulose or chitin. Flavobacterium multivorum which was originally described from human clinical sources can be readily isolated from soil by the dextran enrichment technique.