c-type cytochromes and manganese oxidation in Pseudomonas putida MnB1

Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.     

Cloning and characterization of the fmt gene which affects the methicillin resistance level and autolysis in the presence of triton X-100 in methicillin-resistant Staphylococcus aureus

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.     

Truncation of peptide deformylase reduces the growth rate and stabilizes solvent production in Clostridium beijerinckii NCIMB 8052

The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the ability to form solvents) after prolonged periods of laboratory culture. Several Tn1545 mutants of this organism showing enhanced long-term stability of solvent production were isolated. Four of them harbor identical insertions within the fms (def) gene, which encodes peptide deformylase (PDF). The C. beijerinckii fms gene product contains four diagnostic residues involved in the Zn2+ coordination and catalysis found in all PDFs, but it is unusually small, because it lacks the dispensable disordered C-terminal domain. Unlike previously characterized PDFs from Escherichia coli and Thermus thermophilus, the C. beijerinckii PDF can apparently tolerate N-terminal truncation. The Tn1545 insertion in the mutants is at a site corresponding to residue 15 of the predicted gene product. This probably removes 23 N-terminal residues from PDF, leaving a 116-residue protein. The mutant PDF retains at least partial function, and it complements an fms(Ts) strain of E. coli. Northern hybridizations indicate that the mutant gene is actively transcribed in C. beijerinckii. This can only occur from a previously unsuspected, outwardly directed promoter located close to the right end of Tn1545. The Tn1545 insertion in fms causes a reduction in the growth rate of C. beijerinckii, and, associated with this, the bacteria display an enhanced stability of solvent production. The latter phenotype can be mimicked in the wild type by reducing the growth rate. Therefore, the observed amelioration of degeneration in the mutants is probably due to their reduced growth rates.     

In situ analysis of adhesion molecule expression in kidneys infected with murine malaria

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P(ow) 2.8) but they could grow in medium overlaid with organic solvents having a log P(ow) value higher than that of toluene such as p-xylene (log P(ow) 3.1), cyclohexane (log P(ow) 3.4) and n-hexane (log P(ow) 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3.7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.     

Second site mutations specifically suppress the Fix- phenotype of Rhizobium meliloti ndvF mutations on alfalfa: identification of a conditional ndvF-dependent mucoid colony phenotype

Rhizobium meliloti mutants carrying ndvF insertion or deletion mutations induce nodules on alfalfa which contain very few infected cells and fail to fix N2 (Fix-). We have characterized five independent second site mutations (designated sfx) which completely suppress the Fix- phenotype of ndvF mutants on Medicago sativa but not on another R. meliloti host Melilotus alba. Genetic mapping and phenotypic analysis revealed that the suppressor mutations sfx-1, sfx-4 and sfx-5 mapped to a single locus which was distinct from another locus defined by the sfx-2 and sfx-3 mutations. Tn5-mob-mediated conjugal mapping experiments showed that the sfx-1 locus was located clockwise from trp-33 on the R. meliloti chromosome and a detailed cotransduction map of this region was generated. To clone the sfx-1 locus, we prepared a cosmid library from total DNA obtained from an sfx-1, ndvF deletion strain. From this library, a cosmid pTH56, which converted Fix- ndvF mutants to Fix+, was isolated. Southern blot analysis provided direct physical evidence that the insert DNA in plasmid pTH56 was contiguous with the sfx-1 region. On low osmolarity glutamate-yeast extract-mannitol-salts medium (GYM) agar medium, ndvF insertion and deletion mutants were found to have a mucoid colony phenotype, as opposed to the dry colony phenotype of the wild-type strain. This phenotype was shown to be dependent on the exoB and expE genes required for synthesis of exopolysaccharide II in R. meliloti but not to be dependent on genes required exclusively for the synthesis of the succinoglycan or exopolysaccharide I. Transduction of either sfx-1 or sfx-2 or transfer of the cosmid pTH56 into the ndvF mutants restored them to a wild-type dry colony phenotype. The mucoid phenotype is not responsible for the Fix- phenotype of ndvF mutants as the Fix-, ndvF exp double mutants can be complemented to Fix+ by introducing plasmids which carry only the wild-type ndvF genes.     

Fluorescent pseudomonad pyoverdines bind and oxidize ferrous ion

Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe2+ -pyoverdine than for Fe3+ -pyoverdine. At pH 7.4, about 90% of Fe3+ was bound by pyoverdine Pa-C after 24 h whereas Fe2+ was bound by the pyoverdine completely in only 5 min. The possibility that Fe2+ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe2+ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe2+ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe3+ -specific chelating agent, resulted in the formation of a Fe3+ -hydroxyquinoline complex, suggesting that the iron in the Fe2+ -pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.     

A chaperone in the HSP70 family controls production of extracellular fibrils in Myxococcus xanthus

Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.     

Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria

A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.     

Impaired cerebellar long-term potentiation in type I adenylyl cyclase mutant mice

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.     

Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10(-6)). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used Tn5 mutagenesis to generate a PS- mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycI. The Tn5 insertion in TP39 is approximately 1.5 kb upstream of cycI, and our results show that it is polar onto cycI. The cycI gene has been physically mapped to a region of chromosome I that is approximately 700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.     

New derivatives of TOL plasmid pWW0

Two new segregants, PPW1-1 and PPW161-1, of Pseudomonas putida were isolated from the stock cultures PaW85(pWW0) and PaW85(pWW0-161). Strain PPW1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. A deletion of the left-hand of transposon Tn4651, including the upper-operon genes, had taken place in plasmid pWW0mut1, isolated from strain PPW1-1. Additional deletions were observed in pWW0mut1 after 'benzoate-curing' (plasmids pWW0mut15, pWW0mut19, pWW0mut27). The genes of the upper-operon and beginning of the meta-operon were deleted from pWW0-161mut1, isolated from strain PPW161-1. Despite this deletion, cells of PPW161-1 grew on all normal TOL plasmid substrates. The Tol+ phenotype was stable in cells of PPW161-1 growing on benzoate. We propose that this is because in cells of strain PPW161-1 the catabolic genes deleted from pWW0-161mut1 were integrated into the chromosome at the site where the (chromosomally encoded) ortho-pathway genes are located, resulting in the inability of the cells to use this pathway.     

Genetic analysis of the chitinase system of Serratia marcescens 2170

To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.     

Tn10-mediated inversions fuse uridine phosphorylase (udp) and rRNA genes of Escherichia coli

Two strains carrying metE::Tn10 insertions (upstream of the udp gene) were used to isolate mutants of Escherichia coli overexpressing udp. These strains differ in their gene order; one contains an inversion between the rrnD and rrnE rRNA operons. Selection was based on the ability of overexpressed Udp to complement thymine auxotrophy. Chromosomal rearrangements that connect the udp gene and promoters of different rrn operons were obtained by this selection. Seven of 14 independent mutants selected in one of the initial strains contained similar inversions of the metE-rrnD segment of the chromosome (about 12% of its length). Another mutant contained traces of a more complicated event, inversion between rrnB and rrnG operons, which was followed by reinversion of the segment between metE and the hybrid rrnG/B operon. Similar inversions (udp-rrn) in a strain already carrying an rrnE-rrnD inversion flip the chromosomal segment between metE and rrnD/E in the opposite direction. In this case, inversions are also accompanied by duplications of the chromosomal region between the rrnA and hybrid udp-rrnD/E operons. PCR amplification with a set of oligonucleotides from the rrn, Tn5, and met genes was used for more detailed mapping. Amplified fragments of the rearranged chromosomes connecting rrnD sequences and insertion elements were sequenced, and inversion endpoints were established.