Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.     

A method for the construction of in frame substitutions in operons: deletion of the essential Escherichia coli holB gene coding for a subunit of the DNA polymerase III holoenzyme

To investigate the putative five-gene operon at 24.9 min on the Escherichia coli genome, which comprises the genes pabC, yceG, tmk, holB and ycfH, a method for the construction of an in frame deletion strain of the essential E. coli holB gene was developed. HolB, also referred to as delta prime or delta', is a subunit of the DNA polymerase III (Pol III) holoenzyme. The holB gene was replaced by the kanamycin resistance gene kka1, coding for amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene was expressed under the control of the promoter(s) of the putative five-gene operon. The holB gene is essential for bacterial growth and the deletion of holB exhibits no polar effects on the adjacent genes tmk or ycfH in terms of cell viability. The method of the holB null construction presented in this work allows for a simplified studying of interactions between the different subunits of DNA polymerase III.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.     

arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon.

In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype). Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d). The Ts- growth phenotype was also shown to be associated with previously described cyd alleles. In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide. Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature. Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background. Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3). Two extragenic suppressors were shown to be null alleles in either arcA or arcB. One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function. Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.     

Cloning and characterization of the Burkholderia vietnamiensis norM gene encoding a multi-drug efflux protein

Polymyxin B-sensitive mutants in Burkholderia vietnamiensis (Burkholderia cepacia genomovar V) were generated with a mini-Tn5 encoding tetracycline resistance. One of the transposon mutants had an insertion in the norM gene encoding a multi-drug efflux protein. Expression of B. vietnamiensis norM in an Escherichia coli acrAB deletion mutant complemented its norfloxacin hypersensitivity, indicating that the protein functions in drug efflux. However, no effect on antibiotic sensitivity other than sensitivity to polymyxin B was observed in the B. vietnamiensis norM mutant. We demonstrate that increased polymyxin sensitivity in B. vietnamiensis was associated with the presence of tetracycline in the growth medium, a phenotype that was partially suppressed by expression of the norM gene.     

The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein.

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.     

Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp. rengei

Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.     

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.     

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.     

Generation of Mini-Tn10 transposon insertion mutant library of Bacillus thuringiensis for the investigation of genes required for its bacteriocin production

Bacillus thuringiensis strain BUPM4 is known for its ability to produce a bacteriocin, called Bacthuricin F4 (BF4), which inhibits the growth of several Gram-positive bacteria and particularly Bacillaceae . This study aimed to use the insertional transposon mutagenesis approach for disrupting and thus identifying genes associated with BF4 synthesis. Here, the mini-Tn 10 transposon was used to generate a library of B. thuringiensis mutants. Twenty thousand clones were screened for the search of mutants with affected bacteriocin synthesis. By molecular hybridization, it was demonstrated that the mini-Tn 10 transposition occurred in different sites. Clone MB1, containing a mini-Tn 10 single-copy insertion, lost the BF4 synthesis, but maintained its immunity to BF4. The flanking sequences surrounding the mini-Tn 10 insertion were cloned and sequenced. Homology searches of the surrounding ORFs revealed a strong similarity to a phage tail component, which allowed us to postulate that BUPM4 bacteriocin could be a phage tail-like one.     

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.