Mu D1 Research Articles

Transposon Mutagenesis of Foodborne Pathogenic Escherichia coli.

The Enterobacteriaceae, and in particular, Escherichia coli including foodborne pathotypes are particularly amenable to transposon mutagenesis. Here we describe the use of mini-Tn5 and Mu d1(Ap lac) to generate transposon inserts for analysis of enterohemorrhagic Escherichia coli EDL933. We also discuss how to array the library in 96-well plates and sequence individual clones for further analysis.

Differential mechanisms of opioidergic and dopaminergic systems of the ventral hippocampus (CA3) in anxiolytic-like behaviors induced by cholestasis in mice

There are several studies carried out to test the effect of cholestasis on memory impairment and anxiolytic-like behaviors. Some previous studies have shown that cholestasis alters the activity of opioidergic and dopaminergic systems. The aim of the present study is however to investigate the role of mu opioid, D1 and D2 dopamine ventral hippocampal (CA3) receptors upon cholestasis-induced anxiolytic-like behaviors in hole-board task. Male mice weighing 25–30g were used. Cholestasis was induced by ligation of the main bile duct. Our data indicated that cholestasis can induce anxiolytic-like response. Furthermore, the results showed that the intra-CA3 injection of naloxone, a mu receptor antagonist at 0.25 and 0.5µg/mouse, SCH23390, a D1 dopamine receptor antagonist or sulpiride, as a D2 dopamine receptor antagonist, 5min before testing, reversed the cholestasis-induced anxiolytic-like behaviors seven days after bile duct ligation (BDL). Unlike the higher dose of SCH23390 (0.5µg/mouse) which induced anxiogenic-like behaviors, other doses of the above drugs did not alter the exploratory behaviors in examined mice. Based on our findings, co-administration of the subthreshold dose of naloxone (0.125µg/mouse), SCH23390 or sulpiride, and SCH23390 with sulpiride, neither altered exploratory behaviors in animals nor reversed the cholestasis-induced anxiolytic-like behaviors, seven days post BDL. Current results demonstrated firstly, the anxiolytic-like behaviors are evident in cholestatic mice seven days post BDL; secondly, there are plausible mechanisms governing the involvement of the CA3 opioidergic and dopaminergic systems in this phenomenon and thirdly, there seem to be no interaction between these systems.

Nac-mediated repression of the serA promoter of Escherichia coli.

Escherichia coli and related bacteria contain two paralogous PII-like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR-type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high-copy plasmids, Nac imparts a growth defect to wild-type cells in an expression level-dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. beta-Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10-fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co-ordinated manner when cells become starved for nitrogen.

Molecular analysis of the aidD6: : Mu dl (bla lac) fusion mutation of Escherichia coli K12

In this report we present genetic and biochemical evidence indicating that the aidD6::Mu d1 (bla lac) fusion is an insertion of Mu d1 (bla lac) into the alkB coding sequence. We describe the phenotypic effects resulting from this mutation and compare them with the effects of alkB22, alkA and ada mutations. We also constructed an alkA alkB double mutant and compared its phenotype with that of the single mutant strains. The observation that the methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistance of the double mutant is approximately at the level predicted from the additive sensitivity of each of the single mutants suggests that these two gene products act in different pathways of DNA repair.

Novel two-component transmembrane transcription control: regulation of iron dicitrate transport in Escherichia coli K-12

Citrate and iron have to enter only the periplasmic space in order to induce the citrate-dependent iron(III) transport system of Escherichia coli. The five transport genes fecABCDE form an operon and are transcribed from fecA to fecE. Two genes, termed fecI and fecR, that mediate induction by iron(III) dicitrate have been identified upstream of fecA. The fecI gene encodes a protein of 173 amino acids (molecular weight, 19,478); the fecR gene encodes a protein of 317 amino acids (molecular weight, 35,529). Chromosomal fecI::Mu d1 mutants were unable to grow with iron(III) dicitrate as the sole iron source and synthesized no FecA outer membrane receptor protein. Growth was restored by transformation with plasmids encoding fecI or fecI and fecR. FecA and beta-galactosidase syntheses under transcription control of the fecB gene (fecB::Mu d1) were constitutive in fecI transformants and were regulated by iron(III) dicitrate in fecI fecR transformants. The amino acid sequence of the FecI protein contains a region close to the carboxy-terminal end for which a helix-turn-helix motif is predicted, which is typical for DNA-binding regulatory proteins. The FecI protein was found in the membrane, and the FecR protein was found in the periplasmic fraction. It is proposed that the FecR protein is the sensor that recognizes iron(III) dicitrate in the periplasm. The FecI protein activates fec gene expression by binding to the fec operator region. In the absence of citrate, FecR inactivates FecI. The lack of sequence homologies to other transmembrane signaling proteins and the location of the two proteins suggest a new type of transmembrane control mechanism.

Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions

Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu d1(lacZbla) were analyzed by sequencing the psi::lacZ(Mu d1) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning. Our DNA sequence analysis showed that the MuR DNA in Mu d1 has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu d1-R. Also, Mu d1s in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu d1 insertions into the genome as a whole. Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu d1) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu d1s. One psiC::lacZ(Mu d1) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu d1)s lie in the gltB gene, and one psiQ::lacZ(Mu d1) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu d1) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase. psiE and psiF::lacZ(Mu d1)s lie in uncharacterized open reading frames near the xylE and phoA genes, respectively. Six other psi::lacZ(Mu d1)s lie in yet unreported Escherichia coli sequences.

Aerobic regulation of the Escherichia coli tonB gene by changes in iron availability and the fur locus

The tonB gene is required for the transport of several different iron-siderophore complexes across the Escherichia coli outer membrane. In this study, transcriptional regulation of the tonB gene was investigated by using three different tonB-lacZ fusions to monitor tonB expression under aerobic conditions and in the presence of a wild-type tonB gene. Prior work by other laboratories suggests that tonB is expressed at low constitutive levels regardless of changes in iron availability or the fur locus. In contrast, these data show that tonB transcription is repressed threefold by growth in the presence of FeCl3 compared with growth in the presence of the iron chelator dipyridyl and that this repression requires the fur locus. A 168-base-pair DNA fragment carrying the tonB promoter was sufficient for the observed transcriptional regulation. In addition, the tonB gene appeared to have a substantially stronger promoter than previously recognized. The inability of other laboratories to detect tonB transcription regulation appears to be due to the extremely slow growth of iron-starved tonB strains and the use of Mu d1(lac Apr)- or lambda plac Mu53-generated fusions that encode a thermolabile TrpA-LacZ hybrid protein. The data also suggest that the previously reported growth phase regulation of tonB occurs only in media with intermediate levels of available iron and is due to iron starvation-induced derepression as the culture approaches stationary phase.

Transcriptional analysis of the flagellar regulon of Salmonella typhimurium

In Salmonella typhimurium, nearly 50 genes are involved in flagellar formation and function and constitute at least 13 different operons. In this study, we examined the transcriptional interaction among the flagellar operons by combined use of Mu d1(Apr Lac) cts62 and Tn10 insertion mutants in the flagellar genes. The results showed that the flagellar operons can be divided into three classes: class I contains only the flhD operon, which is controlled by the cAMP-CAP complex and is required for expression of all of the other flagellar operons; class II contains seven operons, flgA, flgB, flhB, fliA, fliE, fliF, and fliL, which are under control of class I and are required for the expression of class III; class III contains five operons, flgK, fliD fliC, motA, and tar. This ordered cascade of transcription closely parallels the assembly of the flagellar structure. In addition, we found that the fliD defect enhanced expression of the class III operons. This suggests that the fliD gene product may be responsible for repression of the class III operons in the mutants in the class II genes. These results are compared with the cascade model of the flagellar regulon of Escherichia coli proposed previously (Y. Komeda, J. Bacteriol. 170:1575-1581, 1982).

Mutational analysis of the histidine operon promoter of Salmonella typhimurium

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage λpSG1. The level of β-galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased β-galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.

Genetics of the iron dicitrate transport system of Escherichia coli.

Escherichia coli B and K-12 express a citrate-dependent iron(III) transport system for which three structural genes and their arrangement and products have been determined. The fecA gene of E. coli B consists of 2,322 nucleotides and encodes a polypeptide containing a signal sequence of 33 amino acids. The cleavage site was determined by amino acid sequence analysis of the unprocessed protein and the mature protein. For the processed form a length of 741 amino acids was calculated. The mature FecA protein in the outer membrane contains at the N terminus the "TonB box," a pentapeptide, which has hitherto been found in all receptors and colicins which functionally require the TonB protein. In addition, the dyad repeat sequence GAAAATAATTCTTATTTCG is proposed to serve as the binding site of the Fur iron repressor protein. The fecB gene was mapped downstream of fecA and encodes a protein with an apparent molecular weight of 30,000. It was synthesized as a precursor, and the mature form was found in the periplasm. The fecD gene follows fecB and was related to a membrane-bound protein with an apparent molecular weight of 28,000. In Mu d1 insertion mutants upstream of fecA, the fec genes were not inducible by iron limitation and citrate, indicating a regulatory region, termed fecI, which controls fec gene expression.

Unmasking of bacteriophage Mu lipopolysaccharide receptors in Salmonella enteritidis confers sensitivity to Mu and permits Mu mutagenesis

The human pathogen Salmonella enteritidis 3b was found to be highly resistant to phage P22 and Mu derivatives. The Mu sensitivity (musA1) allele from Salmonella typhimurium could be transferred to S. enteritidis 3b at low frequency by cotransduction with hisG::Tn10. Sensitivity to Mu resulted in a large reduction in the number of lipopolysaccharide core-region oligosaccharides that were substituted with O-antigen polysaccharide. The residual high-molecular-weight lipopolysaccharide appeared to be a hybrid displaying O antigens which were immunologically related to those of S. typhimurium and not to those of S. enteritidis. Consequently, Mu d1(Ap lac) could then be transduced into Mus strains forming stable lysogens. On temperature induction, Mu transposition could easily be used to generate mutations in genes coding for cell surface antigens including fimbriae, lipopolysaccharide, and flagella.

Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound

Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction. The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence. No similarity was found with the promoter areas of Rhizobium trifolii, R. meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.

The phoBR operon in Escherichia coli K-12.

The phoB and phoR genes encode a transcription activator and a sensory protein of the phosphate regulon, respectively. It is shown here that they were transcribed as an operon in which the phoB gene was promoter proximal. Although an operon structure was suggested previously (K. Makino, H. Shinagawa, M. Amemura, and A. Nakata, J. Mol. Biol. 190:37-44 and 192:549-556, 1986), previous results showed only that phoR gene expression during phosphate limitation is dependent on the upstream phoB promoter. The phoR gene could still have had its own promoter for expression in the presence of phosphate. Two polar transposon-induced mutations are described which simultaneously abolished phoB and phoR gene function in cis; one mutation mapped in the phoB gene, and the other mapped upstream of the phoB gene. These results demonstrate an operon structure, in which phoR gene function required expression from the phoB promoter. Unexpectedly, an antisense pho omega Mu d1(lacZ) insertion within the promoter-proximal end of the phoB gene expressed the lacZ reporter gene, thus allowing for the possibility that the phoBR operon is regulated by an antisense RNA.

In vivo formation of gene fusions in Pseudomonas putida and construction of versatile broad-host-range vectors for direct subcloning of Mu d1 and Mu d2 fusions.

The Mu d1 and Mu d2 prophages were integrated into the conjugative broad-host-range plasmid R751. The two plasmids were then transferred into Pseudomonas putida, and derivatives carrying intact Mu prophages were recovered. After induction of Mu at 42 degrees C, both operon and gene fusions were observed on 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) plates. Broad-host-range vectors were constructed which allow direct cloning of both operon or gene fusions and their analysis in Escherichia coli and P. putida. By using one of these vectors, two operon fusions were isolated from the P. putida chromosome and comparatively analyzed in E. coli and P. putida.

Cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.

The enteric bacterium Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo only under anaerobic growth conditions. We initiated a genetic analysis of the cobalamin biosynthetic (cob) gene cluster, Stable cob::lac operon fusions were generated by insertions of a transposition-defective derivative of bacteriophage Mu d1 (Ap lac) into the cob genes. beta-Galactosidase synthesis was repressed in the presence of exogenously supplied cyanocobalamin, demonstrating that the cobalamin biosynthetic pathway was regulated by end-product repression. Transcriptional polarity studies showed that the cob genes responsible for synthesis of the corrinoid intermediate cobinamide (branch I of the pathway) were organized into a single operon. Genes for the synthesis of 5,6-dimethylbenzimidazole (branch II) and the final assembly of the complete cobalamin molecule (branch III) were organized into two or more additional operons. All of the known cob genes (in branches I, II, and III) were transcribed in a counterclockwise direction relative to the S. typhimurium genetic map. These genes are located at 41 map units and near the his operon. No essential genes lie between the his and cob operons. Mutants that carried deletions extending from the his genes into the cob region were isolated and characterized. By using these mutants, a deletion map of the branch I cob operon was constructed and the order of genes (his-cobI-cobIII-cobII) was inferred.

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.

Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.

Divergent transcription of the sn-glycerol-3-phosphate active transport (glpT) and anaerobic sn-glycerol-3-phosphate dehydrogenase (glpA glpC glpB) genes of Escherichia coli K-12.

The glpTQ operon and the glpA and glpB genes are located adjacent to one another near min 49 of the linkage map of Escherichia coli K-12. The positions and directions of transcription of the glpA and glpB genes with respect to the glpTQ operon were determined in the present work. Strains harboring Mu d1(Ap lac) fusions in either glpA or glpB were converted to the respective lambda p1(209) lysogens. Induction of these lysogens with mitomycin C resulted in production of Lac+ phage progeny which carried adjacent chromosomal DNA. Genetic crosses with a collection of glpT mutant strains were performed with several such phage lines. A fine-structure deletion map of the glpT gene was thus constructed. All phages used for this mapping carried DNA starting with the promoter-proximal end of glpT. This indicated that the glpTQ operon and the glpA and glpB genes are transcribed divergently. Additional evidence supporting this conclusion was obtained by physical mapping of restriction endonuclease cleavage sites in plasmids carrying these genes and in plasmids carrying glpA-lacZ or glpB-lacZ fusions. A new designation (glpC) for the gene encoding the 41,000-Mr subunit of the anaerobic sn-glycerol-3-phosphate dehydrogenase was proposed to distinguish it from the glpA gene, which encodes the 62,000-Mr subunit of the dehydrogenase, and the glpB gene, which encodes a membrane anchor subunit of the dehydrogenase. These three genes were present in an operon transcribed in the order glpA glpC glpB in the clockwise direction on the linkage map of E. coli.

Catabolite repression of thiosulfate reduction bySalmonella typhimurium

The effect of glucose and other carbon sources on thiosulfate reduction and on the expression ofphs bySalmonella typhimurium was examined. Glucose repressed both H2S production from thiosulfate and methyl viologen-linked thiosulfate reductase activity. Cyclic AMP (2 mM) in the growth medium restored both activities. Cyclic AMP was essential for both activities in acya mutant. Glucose and many other sugars repressedphs expression in both Cya+ and Cya−phs::Mu d1(Aprlac) operon fusion mutants. Increasing cyclic AMP to 10 mM increasedphs expression in the presence of some, but not all, sugars. It appears that catabolite repression of thiosulfate reduction inS. typhimurium involves more than a simple requirement for cyclic AMP.