Identification of Cold Shock Gene Loci in Sinorhizobium meliloti by Using a luxAB Reporter Transposon

Using a luxAB reporter transposon, seven mutants of Sinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15°C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three rrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrn operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene, cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15°C.     

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.     

利用Tn5-1063转座诱变法分离苜蓿中华根瘤菌042BM noeB基因的研究

采用三亲本杂交方法将带有Tn5-1063(含luxAB)的质粒pRL1063a导入苜蓿中华根瘤菌(Sinorhizobium meldoti)042BM,进行转座子插入诱变,在含有氯霉素、卡那霉素的TY平板上筛选接合子。通过结瘤试验,从1000个突变株中,筛选到3个结瘤突变株042BMR5、042BMR11和042BRM29。它们都表现出发光酶活性,表明转座子正向插入到基因组中的某个启动子下游。Southern杂交结果证实,转座子均为单一位点插入。对042BMR5突变株基因组进行反向PCR,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到苜蓿中华根瘤菌的共生质粒pSymA noeB基因内。根据基因组中noeB上游和下游序列扩增出042BM noeB,其与苜蓿中华根瘤菌1021 noeB的同源性为98％,而与NoeB蛋白的氨基酸序列相似性为95％。疏水性分析发现,NoeB是一个跨膜蛋白,在N末端有4个跨膜区,其中包含3个初级螺旋和1个次级螺旋。     

Sinorhizobium meliloti genes involved in tolerance to the antimicrobial peptide protamine

The innate resistance of plants and animals to microbial infection is mediated in part by small cationic peptides with antimicrobial activity. We assessed the susceptibility of the alfalfa symbiont Sinorhizobium meliloti to the model antimicrobial peptide protamine. Twenty-one Tn5-induced mutants showing increased sensitivity to protamine were isolated, and nine were further characterized in detail. These nine mutants carried distinct transposon insertions that affected a total of seven different genes. Three of these genes are involved in exopolysaccharide and beta-(1,2)-glucan biosynthesis (exoT, exoU and ndvB), three other genes are implicated in nitrogen metabolism, such as a putative dyhidropyrimidinase, hutU and ureF, and the last gene exhibited similarity to the ATP binding cassette family of membrane transporters. Symbiotic defects ranging from severe to moderate were displayed by some of the protamine-hypersensitive mutants suggesting that S. meliloti possess active mechanisms to counteract hypothetical cationic peptides that may be produced by its host plant.     

Genetic analysis of tag mutants of Myxococcus xanthus provides evidence for two developmental aggregation systems

Temperature-dependent aggregation mutants (tag) of the myxobacterium Myxococcus xanthus aggregated into mounds and developed into fruiting bodies normally at 28 degrees C; however, they failed to form mounds at 34 degrees C. The timing of sporulation was unaffected by the mutations, and normal numbers of spores were produced at both permissive and restrictive temperatures. This class of mutations was originally identified through screening of ethyl methanesulfonate (EMS)-generated mutations. Subsequent work identified a linked insertion of transposon Tn5, which was used to map the EMS-generated mutations to four loci. In this paper, we describe the cloning of the tag loci and the use of transposon mutagenesis to further analyze the tag loci. Nine tag complementation groups spanning 8.5 kilobase pairs of DNA were identified through mapping of 28 independent Tn5 insertions. All insertion and deletion mutants had the same phenotype as the EMS mutants: they were temperature sensitive for mound formation. This result suggests that M. xanthus has at least two sets of genes for developmental aggregation. The tag genes constitute one set of these genes; they are required for normal development at 34 degrees C but are not required for normal development at 28 degrees C.     

Identification of low-temperature-regulated genes in the fire blight pathogen Erwinia amylovora

Genes involved in pathogenicity of several plant pathogens were shown to be induced at relatively cold temperatures. Loci from the fire blight pathogen Erwinia amylovora (Burrill) induced at 18 degrees C were identified using the miniTn5 transposon that contains the promoterless reporter gene gusA coding for beta-glucuronidase (GUS). Certain mutants (2.7%) expressed GUS predominantly at 18 degrees C on minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. A total of 21 mutants were selected: 19 mutants had a transposon insertion in temperature-dependent genetic loci, with a 2.2- to 6.3-fold induction of gusA gene expression at 18 degrees C, and two mutants with impaired growth at 18 degrees C. Some of these genetic loci encoded (i) proteins implicated in flagella biosynthesis, biotin biosynthesis, multi-drug efflux, and type II secretion protein, and (ii) proteins of unknown function.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum . The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn 5  :: lacZ mutant bank for differential expression of β-galactosidase in the presence of P. ultimum . In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn 5  :: lacZ had inserted into a tRNA^Ile gene, which maps within a ribosomal RNA ( rrn ) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.     

Identification of genes relevant to symbiosis and competitiveness in Sinorhizobium meliloti using signature-tagged mutants

Sinorhizobium meliloti enters an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. In order to identify S. meliloti genes required for symbiosis and competitiveness, a method of signature-tagged mutagenesis was used. Two sets, each consisting of 378 signature-tagged mutants with a known transposon insertion site, were used in an experiment in planta. As a result, 67 mutants showing attenuated symbiotic phenotypes were identified, including most of the exo, fix, and nif mutants in the sets. For 38 mutants in genes previously not described to be involved in competitiveness or symbiosis in S. meliloti, attenuated competitiveness phenotypes were tested individually. A large part of these phenotypes was confirmed. Moreover, additional symbiotic defects were observed for mutants in several novel genes such as infection deficiency phenotypes (ilvI and ilvD2 mutants) or delayed nodulation (pyrE, metA, thiC, thiO, and thiD mutants).     

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.     

Diversity of genome structure in Salmonella enterica serovar Typhi populations

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.     

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.     

Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.     

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.