Sequence and mutational analysis of the common nodBCIJ region of Rhizobium sp. (Oxytropis arctobia) strain N33, a nitrogen-fixing microsymbiont of both arctic and temperate legumes

By heterologous hybridization, we have identified the common nodulation genes nodBCIJ of Rhizobium sp. strain N33 within a 8.2-kb PstI fragment. The nodBCIJ genes are located within a 4,620-bp region which also included a consensus nod box promoter. The four open reading frames coding for the nodBCIJ genes contain 657, 1,353, 915, and 789 nucleotides, respectively. We found that the nodA gene was not adjacent to the nodB gene, unlike the situation in many rhizobia. The DNA of the nodBCIJ genes of Rhizobium sp. strain N33 were found to be homologous to the corresponding genes of other rhizobia except for the 3'-coding region of the nodC gene. The deduced NodC protein was the longest of the rhizobia except Bradyrhizobium japonicum. Tn5 mutagenesis of the common nod region of strain N33 revealed that the nodBC genes were essential for nodulation on their temperate hosts Onobrychis viciifolia and Astragalus cicer. By contrast, mutations in the nodI and nodJ genes produced a Nod+ phenotype with a reduced number of nodules on the temperate hosts. Nodules formed on Onobrychis viciifolia by either nodI or nodJ mutants were approximately 10 times smaller than nodules formed by the wild type strain: this reduction in nodule size was not observed on Astragalus cicer.     

Rhizobium fredii synthesizes an array of lipooligosaccharides, including a novel compound with glucose inserted into the backbone of the molecule

Flavonoid cues from the plant host cause symbiotic, nitrogen-fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1). We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USADA191 and 257DH4 both produce a novel LCO with glucose substituted for N-acetyl-D-glucosamine in the backbone of the molecule.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Dependency of contractile reserve on myocardial blood flow: implications for the assessment of myocardial viability with dobutamine stress echocardiography

Fox- mutants of Anabaena sp. strain PCC 7120 are unable to fix dinitrogen in the presence of oxygen. A fragment of the DNA of Anabaena sp. was cloned by complementation of a spontaneous Fox-, cyanophage-resistant mutant, R56, and characterized. Random insertion of transposon Tn5 delimited the complementing DNA to a 0.6-kb portion of the cloned fragment. Sequencing of this region and flanking DNA showed one complete open reading frame (ORF) similar to the gene rfbP (undecaprenyl-phosphate galactosephosphotransferase) and two partial ORFs similar to genes rfbD (GDP-D-mannose dehydratase) and rfbZ (first mannosyl transferase), all of which are active in the synthesis of the O antigen unit of the lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacteria. In a transposon (Tn5-1087b)-induced, Fox-, cyanophage-resistant mutant, B14, the transposon was found within the same rfbP-like ORF. The three ORFs were insertionally inactivated with the omega cassette (P. Prentki and H. M. Krisch, Gene 29:303-313, 1984) or with Tn5::omega. Only the insertions in the rfbZ- and rfbP-like ORFs led to resistance to cyanophages A-1(L) and A-4(L) and to a Fox- phenotype. Electrophoretic analysis showed that interruption of the rfbZ- and rfbP-like ORFs resulted in a change in or loss of the characteristic pattern of the lengths of the LPS, whereas interruption of the rfbD-like ORF merely changed the distribution of the lengths of the LPS to one with a greater prevalence of low molecular weights. According to electron microscopy, interruption of the rfbP-like ORF may have led to aberrant deposition of the layers of the heterocyst envelope, resulting in increased leakage of oxygen into the heterocyst. The results suggest that modified LPS may prevent cyanophage infection of Anabaena sp. vegetative cells and the formation of a functional heterocyst envelope.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

Myxococcus xanthus sasN encodes a regulator that prevents developmental gene expression during growth

Myxococcus xanthus multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations (sasB5, -14, -15, -16, and -17) can bypass the starvation and high-cell-density requirements for expression of the 4521 developmental reporter gene. These mutants express 4521 at high levels during growth and development in an asgB background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the sasB locus cloned from the wild-type chromosome restored the SasB+ phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed sasN-null mutation and Omega4521 Tn5lac in an otherwise wild-type background expressed 4521 at a high level during growth and development. A similar sasN-null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type sasN gene product is necessary for normal M. xanthus fruiting body development and functions as a critical regulator that prevents 4521 expression during growth.     

Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanXB dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

An in vitro investigation into the wear effects of unglazed, glazed, and polished porcelain on human enamel

A pBR322-based vector, pCI195, containing a 4.2-kb region of the conjugative transposon Tn919 was used as a vector for gene cloning in Clostridium difficile. The plasmid was found to integrate into the chromosome of a Bacillus subtilis strain that contained Tn916 delta E. Southern blot analysis of the recombinant demonstrated that pCI195 had inserted into Tn916 delta E by a recombination event. The transposon::plasmid structure could be transferred, by filter mating, from B. subtilis to C. difficile (at a frequency of 10(-8) per donor), where it entered the chromosome at a specific site. Segregation of plasmid and transposon markers was observed on transfer, although the Tn916 delta E::pCI195 was stably maintained in C. difficile. To demonstrate that pCI195 could be used for gene cloning in C. difficile, a 1.1-kb fragment of the C. difficile toxin B gene was cloned into pCI195 to generate pPPM100. Tn916 delta E::pPPM100 was transferred into a nontoxigenic C. difficile strain by filter mating, where it entered the genome at a specific site. pCI195 should be useful as a general cloning vector for C. difficile, as the transposon::plasmid structure could be transferred to different C. difficile strains. This is the first report of gene cloning in C. difficile.     

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.     

Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.     

Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.     

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.     

Electrophoretic behavior and size distribution of the acidic polysaccharides produced by the bacteria Bradyrhizobium (Chamaecytisus) strain BGA-1 and Bradyrhizobium japonicum USDA 110

The electrophoretic behavior in polyacrylamide gels of the acidic polysaccharides produced by the soil bacteria Bradyrhizobium (Chamaecytisus) strain BGA1 and Bradyrhizobiumjaponicum USDA1 10 has been studied. Both polysaccharides were polydisperse, producing a ladder-like pattern after fixation with Alcian Blue and silver staining of the gel. The polysaccharide molecules were separated according to their size, and they behaved as a collection of flexible random coils of different size and similar charge/mass ratio. The electrophoretic behavior was not affected by the presence of acetyl groups in the polysaccharide. The range of molecular weights of the exopolysaccharide produced by B. japonicum USDA110 was wider and with larger molecules than that of the polysaccharide produced by strain BGA1. The resolution was dependent on the electrophoresis buffer; the best results were achieved with Tris-borate; in Tris-glycine buffer, the resolution was worse, and it was not improved by the addition of sodium dodecyl sulfate (SDS).     

Antibiotic production by Erwinia herbicola Eh1087: its role in inhibition of Erwinia amylovora and partial characterization of antibiotic biosynthesis genes

Mutants of Erwinia herbicola Eh1087 (Ant-), which did not produce antibiotic activity against Erwinia amylovora, the fire blight pathogen, were selected after TnphoA mutagenesis. In immature pear fruit Ant- mutants grew at the same rate as wild-type strain Eh1087 but did not suppress development of the disease caused by E. amylovora. These results indicated that antibiosis plays an important role in the suppression of disease by strain Eh1087. All of the Ant- mutations obtained were located in a 2.2-kb region on a 200-kb indigenous plasmid. Sequence analysis of the mutated DNA region resulted in identification of six open reading frames, designated ORF1 through ORF6, four of which were essential to antibiotic expression. One gene was identified as a gene which encodes a translocase protein which is probably involved in antibiotic secretion. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasmid proteins produced in Escherichia coli minicells confirmed the presence of proteins whose sizes corresponded to the sizes of the predicted open reading frame products.