Peptidoglycan Fine Structure of the Radiotolerant Bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)₂ bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear ²⁵²californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H⁺ and Na⁺ ions and in the negative ion mode with H⁻ and CN⁻ ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. ​(Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table ​Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. ​Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. ​(Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)₂ bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A₂₀₄ of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.

TABLE 1

Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radiodurans

Molecular and Biochemical Characterization of the Protein Template Controlling Biosynthesis of the Lipopeptide Lichenysin

Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains of Bacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Three large open reading frames coding for peptide synthetases, designated licA, licB (three modules each), and licC (one module), could be detected, followed by a gene, licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed in Escherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequence l-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile, with minor Leu and Val substitutions at the seventh position.Many strains of Bacillus are known to produce lipopeptides with remarkable surface-active properties (11). The most prominent of these powerful lipopeptides is surfactin from Bacillus subtilis (1). Surfactin is an acylated cyclic heptapeptide that reduces the surface tension of water from 72 to 27 mN m⁻¹ even in a concentration below 0.05% and shows some antibacterial and antifungal activities (1). Some B. subtilis strains are also known to produce other, structurally related lipoheptapeptides (Table ​(Table1),1), like iturin (32, 34) and bacillomycin (3, 27, 30), or the lipodecapeptides fengycin (50) and plipastatin (29).

TABLE 1

Lipoheptapeptide antibiotics of Bacillus spp.

The Atrazine Catabolism Genes atzABC Are Widespread and Highly Conserved

Pseudomonas strain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genes atzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous to atzA, -B, and -C. The sequence identities of the atz genes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- 1,3,5-triazine] is a herbicide used for controlling broad-leaf and grassy weeds and is relatively persistent in soils (51). Atrazine and other s-triazine compounds have been detected in ground and surface waters at levels exceeding the Environmental Protection Agency’s maximum contaminant level of 3 ppb (30).Microbial populations exposed to synthetic chlorinated compounds, such as atrazine, often respond by producing enzymes that degrade these molecules. Most of our current understanding of the genes and enzymes involved in atrazine degradation derives from studies using Pseudomonas strain ADP, in which the first three enzymatic steps in atrazine degradation have been defined (6, 14, 15, 48). The genes atz A, -B, and -C, which encode these enzymes, have been cloned and sequenced. Atrazine chlorohydrolase (AtzA), hydroxyatrazine ethylaminohydrolase (AtzB), and N-isopropylammelide isopropylaminohydrolase (AtzC) sequentially convert atrazine to cyanuric acid (6, 14, 15, 48) (Fig. ​(Fig.1).1). Cyanuric acid and related compounds are catabolized by many soil bacteria (10, 11, 17, 24, 26, 61), and by Pseudomonas sp. ADP, to carbon dioxide and ammonia (35). This provides the evolutionary pressure for the atzA, -B, and -C genes to permit bacterial growth on the more than one billion pounds of atrazine that have been applied to soils globally (20). Here we used a knowledge of the atzA, -B, and -C gene sequences to investigate the presence of homologous genes in other atrazine-degrading bacteria. In this study, we report that five atrazine-degrading microorganisms, which were recently isolated from geographically separated sites exposed to atrazine, contained nearly identical atzA, -B, and -C genes. Open in a separate windowFIG. 1Pathway for atrazine catabolism to cyanuric acid in Pseudomonas sp. strain ADP.

Atrazine-catabolizing bacteria used in this study.

Until recently, attempts at isolating bacteria (18) or fungi (27) that completely degrade atrazine to carbon dioxide, ammonia, and chloride were unsuccessful. While several microorganisms were shown to dealkylate atrazine, they were unable to displace the chlorine atom (41, 54). Since 1994, several research groups have independently isolated atrazine-degrading bacteria that displaced the chlorine atom and mineralized atrazine (3, 7, 13, 35, 39, 46). Six of these bacterial cultures, listed in Table ​Table1,1, were studied here, and the Clavibacter strain had been investigated previously (13).

TABLE 1

Recently isolated atrazine-catabolizing bacteria

Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition

The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.Integrons are DNA elements that capture genes, especially antibiotic resistance genes, by a site-specific recombination system (32). The recombination system consists of a DNA integrase (Int) and two types of recombination sites, attI and attC (59-base element). The integrase gene (int) is located in the 5′ conserved segment of the integron structure (Fig. ​(Fig.1)1) and is a member of the tyrosine recombinase family (1, 4, 13, 23, 24). Three types of integrases, sharing around 50% identity among themselves, have been identified; they define integron classes 1, 2, and 3 (30). The 5′ conserved segment found in class 1 integrons also contains a promoter region responsible for the expression of inserted cassettes (11, 21) and the recombination site attI1 (31). The 3′ conserved segment of the class 1 integrons includes an ethidium bromide resistance determinant (qacEΔ1), a sulfonamide resistance gene (sulI), an open reading frame (ORF5) of unknown function, and further sequences that differ from one integron to another (5, 6, 28). The 3′ conserved segment of class 2 integrons includes transposition genes (20) while that of class 3 integrons has not yet been studied (2). The variable region, located between the two conserved segments, usually contains antibiotic resistance genes; In0 contains no inserted genes while In21 possesses eight cassettes with ten genes (or ORFs) in this region (5, 16). These genes are part of mobile cassettes which include a recombination site, attC, that differs from one gene to another (18, 33). Incoming genes must be associated with an attC to be recognized by the integron integrase and are preferentially inserted at the recombination site attI1 (11). Cassettes are excised as circular intermediates and integrated at core sites by the action of the integrase (8–10). The core site, defined as GTTRRRY, makes up the 3′ end of attI1 and attC, with the crossover taking place between the G and the first T (19). Antibiotic selection pressure can reveal cassette rearrangements in which a given resistance is nearest the promoter and thus most strongly expressed (10). Open in a separate windowFIG. 1General structure of class 1 integrons. Cassettes are inserted in the integron variable region by a site-specific recombination mechanism. The attI1 site is shown by a black circle, core sites are represented by ovals, the attC site is indicated by a black rectangle, and promoters are denoted by P. intIl, integrase gene; qacEΔ1, antiseptic resistance gene; sulI, sulfonamide resistance gene; orf5, gene of unknown function.Site-specific recombination, unlike homologous recombination, is characterized by relatively short, specific DNA sequences and requires only limited homology of the recombining partners (12). Site-specific recombination is an entirely conservative process since all DNA strands that are broken (two per exchange site) are rejoined in a process that involves neither ATP nor DNA synthesis. Homology alignments of site-specific recombinases assign them to two families: the resolvase family, named after the TnpR proteins encoded by the transposons γδ and Tn3, and the integrase family. The integrase family includes over 140 members to date, but they are highly diversified proteins (13, 23). Members of this family, which include the well-studied λ integrase, recombine DNA duplexes by executing two consecutive strand breakage and rejoining steps and a topoisomerization of the reactants. The first pair of exchanges form a four-way Holliday junction and the second pair resolve the junction to complete the recombination. The integrase nucleophile is a conserved tyrosine that becomes associated with a phosphate group on DNA. The cleavage sites on each DNA duplex are separated by 6 to 8 bp with a 5′ stagger, and the tyrosine joins to the 3′ phosphate (17).The initial definition of the integrase family was based on comparisons of seven sequences, and three invariant residues were identified: an HXXR cluster and a Y residue (4). Alignment of 28 sequences identified a fourth invariant position, occupied by an arginine residue (1). These four conserved residues are found in two boxes located in the second half of the protein. A recent analysis has shown that the conserved histidine is present in 136 of the 147 members (93%); this residue is then not conserved in all members of the family (13). Another recent analysis has identified three patches of residues located around box I, which seem to be important in the secondary structure of these proteins (23). In this study, we analyzed the properties of several mutants of the conserved residues R146, R280, and Y312 of the integron integrase IntI1 in in vivo recombination and in vitro substrate binding.

Construction of plasmids overexpressing mutant MBP-IntI1 fusion proteins.

The plasmids encoding various mutants of MBP-IntI1 were constructed by PCR using pLQ369 (50 ng) as a template (15). Two primer pairs, designed with the OLIGO software package (version 4.1; National Biosciences, Plymouth, Minn.), were used to construct each set of mutants. The R146 mutants were constructed with an XcmI-BamHI primer pair [IntI1(R146)-XcmI, 5′-TTCACCAGCTTCTGTATGGAACGGGCATG(A/G)(A/T)AATCAG-3′; IntI1(R146)-BamHI, 5′-CCGGATCCCTACCTCTCACT-3′], the R280 mutants were constructed with an NruI-XmnI primer pair [IntI1(R280)-NruI, 5′-AGCCGTCGCGAACGAGTGC(C/T)(C/T)GAGGG-3′; IntI1(R280)-XmnI, 5′-ACCCCTAATGAAGTGGTTCGTATCC-3′], and the Y312 mutants were constructed with a AatII-ScaI primer pair [IntI1(Y312)-AatII, 5′-ATTCCGACGTCTCTACTACGATGATTT(C/T)CACGC-3′; pLQ369-ScaI, 5′-ATGCTTTTCTGTGACTGGTG-3′] (restriction sites within primer sequences are underlined). PCR conditions were 10 min at 94°C, three cycles consisting of 45 s at 94°C, 45 s at 47°C, and 90 s at 72°C, 30 cycles consisting of 45 s at 94°C, 45 s at 60°C (50°C for Y312 mutants), and 90 s at 72°C, and a final elongation step of 10 min at 72°C. The XcmI, NruI, and AatII primers were degenerate in one or two positions, so that a single primer could give all mutants. Mutant PCR fragments were digested and cloned directly into pLQ369 digested with the same enzymes, except for the R146 mutant fragments that were subcloned into pLQ364 at first. New mutant PCR fragments were then amplified on these subclones, using IntI1(R146)-BamHI and IntI1(R280)-XmnI primers. These mutant PCR fragments were cleaved with BamHI and XmnI, and the resulting fragments were cloned into pLQ369. This avoids the necessity of partial digestion of pLQ369 with XcmI. Mutant clones were digested with restriction endonucleases and sequenced to determine the mutation.

In vivo recombination.

Mutant MBP-IntI1 clones were introduced into Escherichia coli TB1 {F′ araΔ(lac-proAB) rpsL (Str^r) [φ80dlacΔ(lacZ)M15] hsdR(r_K⁻m_K⁻)} containing pLQ428 by transformation (Fig. ​(Fig.22 and Table ​Table1).1). E. coli TB1 cells containing pLQ428 and one of the MBP-IntI1 mutants were grown at 37°C for 3 h in Luria-Bertani medium. Excision of the aacA1-ORFG and/or ORFH cassettes was induced by the overexpression of the malE-intI1 gene by using 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma Chemical Co.) and by incubation at 37°C for another 3 h. Cell culture was done in the presence of 50 μg of ampicillin per ml, 15 μg of amikacin per ml, and 50 μg of chloramphenicol per ml. Plasmid DNA was then prepared from 5-ml cultures with the Perfect Prep DNA extraction kit (Mandel Corporation). In order to determine the capacity of mutant MBP-IntI1 proteins to excise aacA1-ORFG and/or ORFH cassettes of In21, we used PCR primers pACYC184-5′ (5′-TGTAGCACCTGAAGTCAGCC-3′) and pACYC184-3′ (5′-ATACCCACGCCGAAACAAG-3′) (Fig. ​(Fig.2,2, primers 1 and 2) to detect the reduction of pLQ428 length. PCR conditions were 10 min at 94°C, 30 cycles consisting of 1 min at 94°C, 1 min at 60°C, and 5 min at 72°C, and a final elongation step of 10 min at 72°C. A major PCR fragment can be seen in each lane containing a DNA preparation from a mutant clone (Fig. ​(Fig.3,3, lanes 2 to 9). This band is 2,499 bp long and, as determined by restriction enzyme digestions, represents the pLQ428 clone without any cassette excision (data not shown). This band is also observed in the negative control, which is the pMAL-c2 vector without any gene fused to malE (Fig. ​(Fig.3,3, lane 12). Open in a separate windowFIG. 2Representation of plasmids used in this study. The positions of the three invariant residues of the integrase family are indicated, along with restriction sites used to construct mutant proteins. Core sites are represented by black circles, and attCs are shown by white boxes. The numbered arrows represent the PCR primers used to detect excision events, pACYC184-5′ (1) and pACYC184-3′ (2). bla, gene encoding β-lactamase; cat, gene encoding chloramphenicol acetyltransferase; intIl, gene encoding the integron integrase (IntI1); malE, gene encoding the maltose binding protein (MBP); ori, origin of replication; P_tac, tac promoter; P_tet, tetracycline promoter. Only relevant restriction sites are indicated.

TABLE 1

Plasmids used in this study

Heterosexual Transmission of a Murine AIDS Virus

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.Human immunodeficiency virus (HIV) infection is now pandemic. In many countries, HIV has been spread mainly by heterosexual transmission (3, 5). For the prevention of HIV infection, as well as for the development of vaccines against HIV, it is of a great importance to understand the mechanisms of the heterosexual transmission of retroviruses. Since it is difficult to investigate the mechanisms of heterosexual transmission of HIV in humans experimentally, an animal model with a retrovirus which induces an acquired immunodeficiency syndrome like human AIDS would be useful. A murine leukemia virus mixture called LP-BM5 MuLV induces a severe acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice (10). The mixture includes a replication-competent ecotropic virus, mink cell focus-inducing virus, and a replication-defective virus which has been considered to be involved in the pathogenesis of MAIDS (4). With many similarities to human AIDS patients, mice infected with the LP-BM5 MuLV mixture develop splenomegaly, systemic lymphadenopathy, and severe immunodeficiency (4, 11). We previously reported that maternal transmission of LP-BM5 MuLV occurs via mother’s milk with high frequency (12). In the present study, we demonstrate that the heterosexual transmission of LP-BM5 MuLV also occurs with high frequency via genital organs.C57BL/10 (B10) mice were purchased from Japan SLC Inc., Shizuoka, Japan. All mice were specific-pathogen free and were housed in an air-conditioned room. They were given autoclaved water and sterilized pelleted feed. An SC-1 clone chronically infected with LP-BM5 MuLV, the G6 cell line, was kindly supplied by H. C. Morse III, National Institutes of Health, Bethesda, Md. Virus was prepared from the supernatant of G6 cells as previously described (12). The virus preparation was stored at −70°C until use. B10 mice were inoculated by the intraperitoneal route with 0.3 ml of the LP-BM5 MuLV preparation. To increase the frequency of sexual contacts and to avoid pregnancy in the female mice, all male mice were sterilized by vasectomy under anesthesia with pentobarbital (Nembutal). The vasectomized male mice were mated with female mice at least 4 weeks postoperation, since sperm are usually kept alive for 2 to 3 weeks in spermiducts. Excised genital organs were crushed with plastic sticks in 1 ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% sodium dodecyl sulfate, and proteinase K (0.5 mg/ml). Spleen cells were lysed after hemolysis with 0.83% NH₄Cl. Lysed samples were incubated at 50°C for 3 h. DNA was extracted three times with phenol-chloroform, precipitated with cold ethanol, treated with RNase and proteinase K, and dissolved in 0.1 ml of H₂O. LP-BM5 MuLV defective virus genome was detected by Southern blot hybridization combined with PCR as described previously (12). In brief, template DNAs (1 μg per tube) were added to a cocktail adjusted to final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 200 μM deoxynucleoside triphosphate, 100 pmol of each primer (5′-CCTCTTCCTTTATCGACACT-3′ [sense] and 5′-ATTAGGGGGGGAATAGCTCG-3′ [antisense]), and 2 U of Taq DNA polymerase (Boehringer Mannheim) in a total volume of 100 μl and were subjected to 32 cycles of amplification. In each cycle of PCR, the mixture was denatured at 95°C for 1 min (5 min for the first cycle), annealed at 55°C for 3 min, and extended at 72°C for 1 min. The PCR-amplified products were subjected to gel electrophoresis (1.5% agarose) and transferred to a Hybond N+ membrane (Amersham) by the alkaline blotting method. Hybridization was achieved with a 5′ ³²P-labeled probe (5′-TGTCAAAGGGACCAGTTAAG-3′) at 45°C overnight in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.5% sodium dodecyl sulfate–100 μg of salmon sperm DNA per ml. Hybridized membranes were washed twice in 2× SSC at 37°C for 10 min and then in 0.5× SSC at 45°C for 30 min. DNA derived from uterine cervices of uninfected mice was used as a negative control. The limit of sensitivity was approximately 10 copies per tube, as assessed by Southern blot analysis with plasmid DNAs (1/10 of the PCR product).Concanavalin A (ConA) was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Responder spleen cells (2 × 10⁵) were cultured with ConA (5 μg/ml) in 96-well flat-bottomed microculture plates in 0.2 ml of culture medium at 37°C in 7.5% CO₂. The culture medium consisted of RPMI 1640 supplemented with 10% fetal calf serum, penicillin (5,000 IU/100 ml), streptomycin (5,000 μg/100 ml), nonessential amino acids, sodium pyruvate (11.0 mg/100 ml), 2-mercaptoethanol (5 × 10⁻⁵ M), and l-glutamine (29.2 mg/100 ml). On day 2, cultures were pulsed with 1 μCi of [³H]thymidine and incubated for an additional 12 to 18 h. Incorporation of [³H]thymidine into responder spleen cells was quantitated by liquid scintillation counting. Determinations were performed in triplicate; standard errors of the means were generally <5% and therefore have not been indicated.As illustrated in Fig. ​Fig.1,1, in order to investigate the heterosexual transmission of LP-BM5 MuLV from male to female mice, normal male mice were inoculated with LP-BM5 MuLV and vasectomized 1 week later. At 5 weeks after virus inoculation, they were mated with uninfected female mice. After 8 weeks of breeding, female mice were sacrificed and their vaginae, cervices uteri, corpora uteri, inguinal lymph nodes, and spleens were removed and stored at −70°C until use. In the opposite direction, to investigate virus transmission from female to male, normal female mice were inoculated with LP-BM5 MuLV and then mated with uninfected, vasectomized male mice as described above. After 8 weeks of breeding, male mice were sacrificed and their penes, prepuces, inguinal lymph nodes and spleens were removed and stored at −70°C until use. Figure ​Figure22 shows the detection by PCR of the LP-BM5 defective virus genome in genital organs and spleens that were taken from mice mated with their virus-infected counterparts. It was demonstrated that although the defective virus genome was detected in both spleens and genital organs in some male mice (2 of 17 [see Table ​Table1]),1]), as shown in Fig. ​Fig.2,2, lanes 3 and 4, the defective virus genome was detected only in the genital organs, not the spleens (Fig. ​(Fig.2,2, lanes 5 and 6), from most of the male mice. In contrast, all of the female mice were positive for defective virus genome only in the genital organs (Fig. ​(Fig.2,2, lanes 1 and 2). None of the mice examined were positive for the virus genome only in the spleens (this issue is discussed below). It should be noted here that the efficacy of PCR amplification, which was measured by experiments using the mixture of genomic DNA and plasmid DNA containing the defective virus, did not differ among the genital organs and spleens. By using the above strategy, the heterosexual transmission of LP-BM5 MuLV was investigated according to the protocol shown in Fig. ​Fig.1.1. Open in a separate windowFIG. 1Experimental design for examination of heterosexual transmission of the MAIDS virus in B10 mice. i.p., intraperitoneal.Open in a separate windowFIG. 2Detection of the LP-BM5 MuLV defective virus genome by PCR in genital organs and spleens. The template DNAs (1 μg) derived from female or male mice which were bred with LP-BM5 MuLV-infected mice were amplified by PCR. Samples were prepared from either female (lanes 1 and 2) or male (lanes 3 to 6) mice. Lanes 1, 3, and 5, spleen; lane 2, uterine cervix; lanes 4 and 6, penis (from two representative male mice). The PCR products (5 μl) were applied to a 1.5% agarose gel and analyzed by Southern blotting with a probe for the defective virus (12).

TABLE 1

Heterosexual transmission of LP-BM5 MuLV

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient.Bacteria capable of mineralizing 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide, are found in many different phylogenetic groups (2, 3, 7, 11, 22, 23). Evidence suggests that numerous variants of 2,4-D catabolic genes exist and that catabolic operons consist of a near-random mixing of these variants (7). Interspecies gene transfer is a well-documented phenomenon (13), and horizontal gene transfer of the 2,4-D-degrading plasmid pJP4 has been shown (3, 5). However, not all 2,4-D catabolic operons are found on plasmids (10, 11, 16, 20). The extent to which other 2,4-D genes have been exchanged in nature is unknown. The aim of this research was to assess the role of horizontal gene transfer in the evolution of 2,4-D-degrading strains. This article summarizes the results of two aspects of this work—the study of the transfer of the entire 2,4-D pathway by using standard mating experiments and a phylogenetic study of the tfdA gene. The tfdA gene codes for an α-ketoglutarate-dependent 2,4-D dioxygenase which converts 2,4-D into 2,4-dichlorophenol and glyoxylate (6). This 861-bp gene was first sequenced from Ralstonia eutropha JMP134 (19). Two more tfdA genes were cloned from chromosomal locations in Burkholderia strain RASC and Burkholderia strain TFD6 (16, 20). These proved to be identical to each other and 78.5% similar to the original. An alignment of the two variants allowed conserved areas to be identified and primers to be designed for the amplification of tfdA-like genes from other sources (24). Sequence analysis of putative tfdA fragments and the small-subunit ribosomal DNA (SSU rDNA) of the strains carrying them allowed us to construct phylogenies of the genes and their hosts and to look for congruency between them.

Mating experiments.

A collection of 2,4-D degraders containing 15 unique strains as determined by genomic fingerprinting (7) was used as a source of donors in a series of mating experiments (Table ​(Table1).1). Burkholderia cepacia D5, lacking the ability to grow on 2,4-D and not hybridizing to any tfd genes, was used as a recipient in mating experiments. Strain D5 contains neomycin phosphotransferase genes (nptII) carried on transposon Tn5 and is resistant to 50 μg each of kanamycin, carbenicillin, and bacitracin per ml. All of the 2,4-D strains used were sensitive to these antibiotics. Filter matings were performed with a donor-to-recipient ratio of 1:10. Colonies which grew on selective medium (500 ppm of 2,4-D in mineral salts agar [MMO] [23] including 50 μg of kanamycin, carbenicillin, and bacitracin per ml) were subjected to further tests. Their ability to catabolize 2,4-D was tested in liquid medium (same composition as that described above).

TABLE 1

2,4-D-degrading strains, geographic origins, and GenBank accession numbers

Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.The antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] (PRN) is produced by many pseudomonads and has broad-spectrum antifungal activity (1, 5, 12–14, 17). PRN has been implicated as an important mechanism of biological control of fungal plant pathogens by several Pseudomonas strains (12–14), including P. fluorescens BL915, from which the prn genes were isolated (10).Tryptophan was identified as the precursor for PRN, based on the feeding of cultures with isotopically labeled and substituted tryptophan (2, 7, 8, 17, 25). Biosynthetic pathways were proposed as early as 1967 (7) and have been refined on the basis of tracer studies and the isolation of intermediates (Fig. ​(Fig.1)1) (2, 8, 17, 19, 23, 25). Recently, Hammer et al. (9) described the cloning and characterization of a 5.8-kb DNA region which encodes the PRN biosynthetic pathway. This DNA region confers the ability to produce PRN when expressed heterologously in Escherichia coli and contains four genes, prnABCD, each of which is required for PRN production. In the research described here, we used mutants in which each of the four genes was disrupted and strains which overexpress the individual genes to elucidate the function of each gene product in PRN biosynthesis. Open in a separate windowFIG. 1Biosynthetic pathways for PRN as proposed by van Pée et al. (23) (A) and by Chang et al. (2) (B). The reactions catalyzed by the PRN biosynthetic enzymes encoded by the prnABCD genes are indicated above the appropriate reaction arrows.

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are described in Table ​Table1.1. Pseudomonas strains were cultured in Luria-Bertani medium at 28°C. Antibiotics, when used, were added at the following concentrations: tetracycline, 30 μg/ml; and kanamycin, 50 μg/ml. The expression vector pPEH14 consists of the P_tac promoter and rrnB ribosomal terminator from pKK223-3 (Pharmacia, Uppsala, Sweden) cloned into the BglII site of the broad-host-range plasmid pRK290 (4). P_tac is a strong constitutive promoter in Pseudomonas (unpublished data). The PRN biosynthetic genes are the coding regions described by Hammer et al. (9). Each coding region was cloned from the translation initiation codon to the stop codon by PCR with restriction sites added to the ends to facilitate cloning. For prnB, the native GTG initiation codon was changed to ATG. The clones were sequenced after PCR.

TABLE 1

Bacterial strains and plasmids used in this study