Iron respiration-driven proton translocation in aerobic bacteria

Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone.     

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 x 10(-7) per recipient after filter mating with E. faecalis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10(-5) per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302, approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 x 10(-6) per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus.     

Transposon Tn916 mutagenesis in Clostridium botulinum.

The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10(-8) to 10(-5) Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindIII chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.     

Transposon Tn916 mutagenesis in Clostridium botulinum.

The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10(-8) to 10(-5) Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindIII chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.     

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob.

RK2::Mu plasmids and transposon Tn5-Mob were used to mobilize the Legionella pneumophila chromosome. Plate matings between L. pneumophila donors that contained RK2::Mu plasmids and auxotrophic recipients yielded recombinants at frequencies ranging from 10(-6) to 10(-7) per recipient for the markers tested. The presence of a Mu insertion in the chromosome of donors that harbored RK2::Mu plasmids increased the frequency of chromosome transfer of certain selected markers as compared with strains that contained RK2::Mu alone. Cotransfer experiments with Mu-containing donors and a thymidine and tryptophan auxotroph failed to reveal any linkage between the thy and trp loci in L. pneumophila. A strain that contained a chromosomal Tn5-Mob insertion and helper plasmid pRK24.4 transferred chromosomal markers at frequencies of 10(-7) per recipient. These findings suggest that RK2::Mu plasmids and Tn5-Mob may be useful for genetic mapping experiments with L. pneumophila.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.     

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.     

Conjugative transfer of the transposon Tn919 to lactic acid bacteria

Abstract The streptococcal transposon Tn 919 was transferred from Streptococcus faecalis GF590 to selected Group N Streptococcus strains and to one strain each of Lactobacillus plantarum and Leuconostoc cremoris , using the filter mating method. An S. lactis MG1363 Rif^r Tc^r transconjugant also acted as a donor, but was less efficient than GF590. Frequencies of transfer varied between 4.0 × 10⁻⁸ and 5.29 × 10⁻⁵ per recipient. Further analysis of S. lactis MG1363 Sm^r Tc^r transconjugants showed that insertion of Tn 919 into the chromosome was site-specific.     

Cloning of a sequence of Aquaspirillum magnetotacticum that complements the aroD gene of Escherichia coli

A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.     

Transposition of Tn1545-delta 3 in the pathogenic Neisseriae: a genetic tool for mutagenesis.

The ability to study the virulence of pathogenic Neisseria spp. has been greatly limited by the absence of genetic tools which allow the construction of defined mutants. We have engineered a transposon system which allows random mutagenesis of the Neisseria genome at relatively high frequency. Tn1545-delta 3 is a 3.4-kb derivative of the gram-positive transposon Tn1545 encoding resistance to kanamycin. Tn1545-delta 3 was subcloned into an erythromycin-resistant derivative of the mobilizable shuttle vector pLES2 to yield the plasmid pMGC20. This latter plasmid was introduced by conjugation from Escherichia coli S17-1 into Neisseria meningitidis 8013N and Neisseria gonorrhoeae 15063G. Kanamycin-resistant 8013N and 15063G transconjugants appeared at frequencies of 10(-5) and 10(-6), respectively. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that, in Neisseria spp., the transposon excised spontaneously from pMGC20 and integrated into chromosomal DNA. Our studies revealed that (i) transposition of Tn1545-delta 3 was in numerous, apparently distinct sites, (ii) in most cases, for each transconjugant a single copy of Tn1545-delta 3 was integrated into the chromosome, and (iii) several passages on selective media did not induce secondary transposition. The kanamycin resistance marker expressed by the transconjugants was subsequently transformed into a parental background without change in the chromosomal location of the transposon. To assess the role of the general recombination system in the transposition of Tn1545-delta 3, the recA gene of N. meningitidis has been cloned and a rec derivative of 8013N has been engineered. Similar results were obtained when this latter strain was used as recipient, suggesting that recA function were not required for Tn1545-delta 3 transposition in N. meningitidis. Transposition with Tn1545-delta 3 may be an important technique for mutagenesis of the pathogenic neisseriae.     

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).     

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).     

Transposon mutagenesis and strain construction in Zymomonas mobilis

Conjugative or mobilizable plasmids carrying the transposable elements Tn5, Tn501 or mini Mu were readily transferred from Escherichia coli donors into Zymomonas mobilis recipients with frequencies depending both on donor and recipient strain used. With the exception of pULB113 (RP4::mini Mu), all foreign plasmids exhibited high instability in Z. mobilis transconjugants under both selective and non-selective conditions. Transposition events and consequent mutagenesis occurred readily in Z. mobilis transconjugant strains, with Tn5 and Tn501 being far less successful than mini Mu. Transposon mutagenesis with the help of mini Mu resulted in the isolation of a large number of independent auxotrophs with polyauxotrophs, cysteine, methionine and isoleucine requiring-isolates being the most frequent. When chromosomal DNA from all these mutants was digested with various restriction enzymes and the resulting restriction patterns were hybridized with a mini Mu probe, the majority of these mutants appeared to have insertions at different sites of the chromosome. Thus, transposon mutagenesis by mini Mu is proven to be a simple and efficient tool for mutant production and the genetic analysis of Z. mobilis.     

Molecular description and industrial potential of Tn6098 conjugative transfer conferring alpha-galactoside metabolism in Lactococcus lactis

A novel 51-kb conjugative transposon of Lactococcus lactis, designated Tn6098, encoding the capacity to utilize α-galactosides such as raffinose and stachyose, was identified and characterized. Alpha-galactosides are a dominant carbon source in many plant-derived foods. Most dairy lactococcus strains are unable to use α-galactosides as a growth substrate, yet many of these strains are known to have beneficial industrial traits. Conjugal transfer of Tn6098 was demonstrated from the plant-derived donor strain L. lactis KF147 to the recipient L. lactis NZ4501, a derivative of the dairy model strain L. lactis MG1363. The integration of Tn6098 into the genome of the recipient strain was confirmed by Illumina sequencing of the transconjugant L. lactis NIZO3921. The molecular structure of the integration site was confirmed by a PCR product spanning the insertion site. A 15-bp direct repeat sequence (TTATACCATAATTAC) is present on either side of Tn6098 in the chromosome of L. lactis KF147. One copy of this sequence is also present in the L. lactis MG1363 chromosome and represents the sole integration site. Phenotypic characterization of all strains showed that the transconjugant has not only acquired the ability to grow well in soy milk, a substrate rich in α-galactosides, but also has retained the flavor-forming capabilities of the recipient strain L. lactis MG1363. This study demonstrates how (induced) conjugation can be used to exploit the beneficial industrial traits of industrial dairy lactic acid bacteria in fermentation of plant-derived substrates.     

Transposon mutagenesis in Acinetobacter calcoaceticus RAG-1.

Molecular genetic studies of Acinetobacter spp. have been greatly limited by the lack of a method for transposon mutagenesis. In this study, a genetically engineered derivative of Tn10, mini-Tn10PttKm, was conjugally transferred in plate matings from Escherichia coli SM10[(lambda pir)(pLOFPttKm)] to Acinetobacter calcoaceticus RAG-1. Transfer frequencies were dependent on mating ratios and varied from 7.9 x 10(-8) to 3.4 x 10(-7) per recipient cell. The 27 lipase-deficient transconjugants which were isolated exhibited several different phenotypes, including gelatinase mutants, esterase mutants, and putative auxotrophs. Southern blot analysis confirmed the insertion of the mini-Tn10PttKm transposon in single, unique sites in five transconjugants. Four of five lipase mutants contained single insertions of mini-Tn10PttKm in the same chromosomal restriction fragments. To our knowledge, this is the first report of the use of a transposon for direct, generalized mutagenesis in Acinetobacter spp.     

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.     

Tn 7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida

Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7-containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the ColE1 replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pRKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7 but can also derive from conservative mechanisms.     

Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis.

A novel, chromosomally located conjugative transposon in Lactococcus lactis, Tn5276, was identified and characterized. It encodes the production of and immunity to nisin, a lanthionine-containing peptide with antimicrobial activity, and the capacity to utilize sucrose via a phosphotransferase system. Conjugal transfer of Tn5276 was demonstrated from L. lactis NIZO R5 to different L. lactis strains and a recombination-deficient mutant. The integration of Tn5276 into the plasmid-free strain MG1614 was analyzed by using probes based on the gene for the nisin precursor (nisA) and the gene for sucrose-6-phosphate hydrolase (sacA). The transposon inserted at various locations in the MG1614 chromosome and showed a preference for orientation-specific insertion into a single target site (designated site 1). By using restriction mapping in combination with field inversion gel electrophoresis and DNA cloning of various parts of the element including its left and right ends, a physical map of the 70-kb Tn5276 was constructed, and the nisA and sacA genes were located. The nucleotide sequences of Tn5276 junctions in donor strain NIZO R5 and in site 1 of an MG1614-derived transconjugant were determined and compared with that of site 1 in recipient strain MG1614. The results show that the A + T-rich ends of Tn5276 are flanked by a direct hexanucleotide repeat in both the donor and the transconjugant but that the element does not contain a clear inverted repeat.     

Mobilization and transfer of Azospirillum lipoferum plasmid by the Tn5-Mob transposon into a plasmid-free Agrobacterium tumefaciens strain

Azospirillum lipoferum 4B harbors five cryptic plasmids. Several suicide plasmids were used to transfer Tn5-Mob to A. lipoferum 4B. Tn5-Mob insertion mutations of this strain could be obtained at frequencies of 10(-8)-10(-7) per recipient cell. One hundred Tn5-Mob A. lipoferum 4B mutants were used in bacterial matings with a plasmid-free Agrobacterium tumefaciens recipient strain. This is the first report of mobilization, transfer, and replication of an Azospirillum plasmid in Agrobacterium tumefaciens. One transconjugant was found which had lost an indigenous plasmid.