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B L Wanner

Publications and source records attributed to B L Wanner.

At least 55 records · Page 3Linked to original sources
PubMed1992

Genes for phosphonate biodegradation in Escherichia coli.

Escherichia coli has a carbon-phosphorus (C-P) lyase with a broad substrate specificity, whose synthesis is induced many hundred fold during phosphate (Pi) limitation. Fourteen genes for phosphonate metabolism comprise the phnC-to-phnP gene cluster: three gene products (PhnC, PhnD, and PhnE) comprise a binding protein-dependent phosphonate transporter, which also transports Pi and phosphate esters; two gene products (PhnF and PhnO) may have a role in gene regulation; and nine gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, PhnM, PhnN, and PhnP) may comprise a C-P lyase enzyme complex. Phosphonate biodegradation via a C-P lyase appears to be limited by the specificity of the PhnCDE transporter and not by the specificity of the C-P lyase. These interpretations are based on results from a combination of molecular genetic and molecular biological studies on phosphonate metabolism in E. coli.

Biodegradation, Environmental↗
PubMed1991

Involvement of the Escherichia coli phn (psiD) gene cluster in assimilation of phosphorus in the form of phosphonates, phosphite, Pi esters, and Pi.

The phn (psiD) gene cluster is induced during Pi limitation and is required for the use of phosphonates (Pn) as a phosphorus (P) source. Twelve independent Pn-negative (Pn-) mutants have lesions in the phn gene cluster which, as determined on the basis of recombination frequencies, is larger than 10 kbp. This distance formed the basis for determining the complete DNA sequence of a 15.6-kbp BamHI fragment, the sequences of which suggested an operon with 17 open reading frames, denoted (in alphabetical order) the phnA to phnQ genes (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990) Ten Pn- lesions lie in the phnD, phnE, phnH, phnJ, phnK, phnO, and phnP genes. We propose a smaller gene cluster with 14 open reading frames, phnC to phnP, which probably encode transporter and regulatory functions, in addition to proteins needed in Pn biodegradation. On the basis of the effects on phosphite (Pt), Pi ester, and Pi use, we propose that PhnC, PhnD, and PhnE constitute a binding protein-dependent Pn transporter which also transports Pt, Pi esters, and Pi. We propose that PhnO has a regulatory role because a phnO lesion affects no biochemical function, except for those due to polarity. Presumably, the 10 other phn gene products mostly act in an enzyme complex needed for breaking the stable carbon-phosphorus bond. Interestingly, all Pn- mutations abolish the use not only of Pn but also of Pt, in which P is in the +3 oxidation state. Therefore, Pn metabolism and Pt metabolism are related, supporting a biochemical mechanism for carbon-phosphorus bond cleavage which involves redox chemistry at the P center. Furthermore, our discovery of Pi-regulated genes for the assimilation of reduced P suggests that a P redox cycle may be important in biology.

DNA Transposable Elements↗
PubMed1990

Molecular biology of carbon-phosphorus bond cleavage. Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B.

Whereas bacteria such as Escherichia coli have been known for some time to cleave carbon-phosphorus (C-P) bonds in unactivated alkylphosphonates, the enzymes responsible for C-P lyase activity have resisted detection or purification. Genes from E. coli B that support growth on alkylphosphonates as the sole phosphorus source have now been cloned (B. L. Wanner and J. A. Boline, unpublished data). Deletion analysis demonstrated that at least 13 kilobases of DNA information is required for E. coli to express the phosphonate utilization phenotype (Phn+). The complete nucleotide sequence of 15,611 bases has been determined, and the gene structures were examined. Seventeen open reading frames (phnA to phnQ) were identified in one transcriptional direction and five open reading frames in the divergent direction. Sequence homology searches identify PhnC, PhnK, PhnL, and, possibly, PhnN proteins as members of nucleotide-binding proteins of the binding protein-dependent transport systems. Candidates for other membrane components and regulatory proteins are also identified. A Pho box-like promoter sequence is also found upstream of the gene cluster starting at phnA, which is consistent with the observation of phosphate regulation of the Phn+ response. Fourteen repetitive extragenic palindromic sequences are found in the phn DNA: 10 exist in the extragenic region between phnA and phnB, two between phnD and phnE, and two between phnK and phnL. An unusual finding is that one of the repetitive extragenic palindromic sequences actually overlaps with the reading frame of the phnE gene.

Amino Acid Sequence↗
PubMed1990

Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli.

The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.

Cloning, Molecular↗
PubMed1990

A phoA structural gene mutation that conditionally affects formation of the enzyme bacterial alkaline phosphatase.

The phoA503 mutant was identified as a mutant that shows a novel phoA regulatory phenotype. The phoA503 allele dramatically reduces the synthesis of bacterial alkaline phosphatase activity during Pi starvation in an otherwise wild-type host and during the logarithmic growth phase in a phoR or phoU background. Near-normal amounts of enzyme activity are found in phoR phoA503 or phoU phoA503 mutants when starved for carbon, nitrogen, or sulfur or during the stationary phase, however. Marker rescue and DNA sequence analysis located the phoA503 mutation to the phoA coding region. It is a C-to-T transition that would cause a substitution of Val for Ala-22 in the mature protein. Transcriptional and translational lacZ fusions to both wild-type and mutant alleles demonstrated that phoA gene expression is unaltered. Also, the mutant protein was secreted and processed as efficiently as the wild type. Furthermore, the subunits appeared to dimerize and to be stable in the periplasm. But, greater than 98% of the dimers were inactive and found exclusively as isozyme 1. An activation of preformed phoA503 dimers occurred during the stationary phase with the concomitant conversion into isozymes 2 and 3. We propose that the phoA503 mutation affects a late stage in the formation of active enzyme. An unknown change when Pi is present during stationary-phase growth leads to formation of active dimers, which is responsible for this new conditional phenotype.

Alkaline Phosphatase↗
PubMed1990

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.

Bacteriophage mu↗
PubMed1988

Molecular cloning of the wild-type phoM operon in Escherichia coli K-12.

A metastable bacterial alkaline phosphatase (Bap) phenotype is seen in phoR mutants, which alternately express a Bap-constitutive or -negative phenotype. The alteration is affected by mutations in the phoM region near 0 min. By molecular cloning of the wild-type phoM operon onto a multicopy plasmid and recombining onto the plasmid the pho-510 mutation that abolishes variation, the phoM operon, rather than some nearby gene, was shown to control variation. Complementation tests indicated that the wild-type phoM allele is dominant to the pho-510 mutation when both are in single copy, but whichever allele is present in higher copy appears as dominant when multicopy plasmids are examined. The alternating phenotypic variation of BAP synthesis was not seen in phoR+ cells with multicopy wild-type phoM plasmids, thus showing that the variation is associated with phoM-dependent Bap expression. The alternation acted at the level of phoA transcription; it was also recA independent. BAP clonal variation is phenotypically similar to Salmonella phase variation, which is controlled by a DNA rearrangement. No evidence was found for a DNA change near the phoM operon that might be responsible for the variable Bap phenotype.

Alkaline Phosphatase↗
PubMed1988

Control of bacterial alkaline phosphatase synthesis and variation in an Escherichia coli K-12 phoR mutant by adenyl cyclase, the cyclic AMP receptor protein, and the phoM operon.

Mutant phoR cells show a clonal variation phenotype with respect to bacterial alkaline phosphatase (BAP) synthesis. BAP clonal variation is characterized by an alternation between a Bap+ and Bap- phenotype. The switching is regulated by the phoM operon and the presence of glucose; the pho-510 mutant form of the phoM operon abolishes both BAP clonal variation and the effect of glucose (B.L. Wanner, J. Bacteriol. 169:900-903, 1987). In this paper we show that a mutation of the adenyl cyclase (cya) and the cyclic AMP receptor protein (crp) gene also abolish BAP clonal variation; either simultaneously reduces the amount of BAP made in phoR mutants. Also, the pho-510 mutation is epistatic; it increases BAP synthesis in delta cya phoR and delta crp phoR mutants. These data are consistent with the wild-type phoM operon having a negative, as well as a positive, regulatory role in gene expression. Furthermore, the data suggest that adenyl cyclase and Crp indirectly regulate BAP synthesis in a phoR mutant via an interaction with the phoM operon or its gene products. However, phoM operon expression was unaffected when tested with phoM operon lacZ transcriptional fusions. In addition, the switching Bap phenotype was not associated with an alternation in phoM operon expression.

Adenylyl Cyclases↗
PubMed1987

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.

Bacterial Proteins↗
PubMed1987

Control of phoR-dependent bacterial alkaline phosphatase clonal variation by the phoM region.

phoR mutants with the wild-type Escherichia coli K-12 Var+ phoM region showed clonal variation of bacterial alkaline phosphatase synthesis, whereas mutants with the pho-510 Var- allele did not. The pho-510 mutation is responsible for the phoR mutant constitutive phenotype and probably arose in E. coli K-12 58F+ after X-ray mutagenesis over 40 years ago. I propose that the phoM region controls a change in state of bacterial alkaline phosphatase synthesis, at least in phoR mutants. Four possible molecular mechanisms for how phoM may act are discussed.

Alkaline Phosphatase↗
PubMed1987

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.

Escherichia coli↗
PubMed1987

Molecular cloning of Mu d(bla lacZ) transcriptional and translational fusions.

The vector pBW2 was made to selectively clone chimeric plasmids with chromosomal Mu d(bla lacZ) transcriptional or translational fusions. It was tetracycline resistant and had the carboxyl-terminal end of bla distal to its PstI site. Because ligation of PstI-digested chromosomal DNA of a Mu d(bla lacZ) insertion with pBW2 restored bla, ampicillin-resistant chimeric plasmids were selectable. These plasmids had the Mu d bla amino terminus and simultaneously acquired other Mu d sequences including lacZ, the chromosomal fusion joint, and the DNA adjacent to the nearest chromosomal PstI site. The plasmid pBW2 was useful in the molecular cloning of several psi and pho::lacZ(Mu d) fusions, as well as chromosomal genes located near Mu d insertions.

Bacteriophage mu↗
PubMed1986

Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12.

New pleiotropic mutants were isolated that express either the phoA, psiE or psiO promoter constitutively and simultaneously alter bacterial alkaline phosphatase regulation, carbon utilization or ultraviolet light sensitivity. To do this, Lac+ mutants were isolated from strains with the appropriate lacZ transcriptional fusions. Over 300 independent mutants were characterized, and all that constitutively express phoA map in phoR, phoU, the phosphate-specific transport system or a new locus called phoF. However, only phoU mutants express both phoA and psiE constitutively. Carbohydrate-utilizing mutants that show constitutive expression of psiE and psiO map in cya, crp and, possibly, crr. Also, numerous ultraviolet-light-sensitive mutants were discovered that show increased psiO expression and map in lon. Some other mutations that lead to constitutive psiO expression (which is normally induced either by phosphate, nitrogen or carbon starvation or anoxia) show decreased expression of phoA. Also, several mutants were found that show an unusual metastable character affecting psiO or phoA transcription. In these, colonies spontaneously switch between an induced and repressed "state" with respect to lac or bacterial alkaline phosphatase expression. In some, the clonal variation of the lactose phenotype or bacterial alkaline phosphatase synthesis is recA-independent and phenotypically resembles phase variation in Salmonella typhimurium. The latter class are called "phase mutants". The mutants are discussed in terms of protein-nucleic acid interactions and/or possible changes in the DNA, i.e. modifications or rearrangements, within the phosphate gene system, that are physiologically regulated.

Alkaline Phosphatase↗
PubMed1986

Bacterial alkaline phosphatase clonal variation in some Escherichia coli K-12 phoR mutant strains.

Several phoR alleles (phoR19, phoR20, phoR68, phoR69, phoR70, and phoR78) led to either a bacterial alkaline phosphatase (BAP)-constitutive phenotype or a variable behavior, depending upon the strain tested. Whereas Escherichia coli K10, MC1000, and XPh4 phoR mutants were constitutive, AB1157, BD792, MC4100, and W3110 phoR mutants displayed the metastable character. For the latter strains, constitutive mutants regularly segregated BAP-negative clones which yielded constitutive variants again at a high frequency. Indeed, the pattern of variation observed in BAP-variable phoR strains is phenotypically analogous to phase variation of the H1/H2 flagellum antigen type in Salmonella typhimurium and the molecular switch between the immune and sensitive states in bacteriophage lambda. The metastable behavior was not a general property of BAP-constitutive mutants, since several phosphate-specific transport-phoU mutations led to a constitutive (stable) phenotype regardless of the strain tested. But in phoR phosphate-specific transport-phoU mutants, the metastable character was epistatic (dominant), and such double mutants showed clonal variation in BAP-variable strains.

Alkaline Phosphatase↗
PubMed1983

Overlapping and separate controls on the phosphate regulon in Escherichia coli K12.

The physiological and genetic controls operating on phosphate-regulated promoters were studied in greater detail. This was done by defining the control for three phosphate-regulated genes: phoA, psiE, and psiO. Each is highly inducible by phosphate starvation. Individually, these phosphate-starvation-inducible, psi, genes at the same time show common and differing features in their molecular control. The phoA gene, encoding alkaline phosphatase, is specifically induced by phosphate starvation. It is negatively controlled by phoR as well as by the phosphate-specific transport (PST) system in Escherichia coli. phoA induction is positively controlled by the phoB, M, and R products; it is unaffected by the cAMP and CAP system. The psiE and psiO genes were studied by using strains with lacZ fused to their respective promoters. psiE-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth. Genetically, psiE-lacZ induction is partially phoB and phoR-dependent. However, its expression is phoM-independent. This implies that phoB/phoR coupled control differs from phoB/phoM coupled control. Repression of psiE-lacZ is substantially altered in only some PST mutants, such as phoT. In addition, psiE-lacZ is negatively controlled by the cAMP and CAP system. psiO-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth or by anaerobiosis. Its expression is unaffected by any pho mutation that has been previously described. A cell density-dependent induction of psiO-lacZ is observed in lon mutants. Also, psiO-lacZ is negatively controlled by the cAMP-CAP system. In summary, these results demonstrate that co-ordinately regulated promoters can have some common regulatory elements while, at the same time, not sharing other controlling factors.

Alkaline Phosphatase↗
PubMed1982

Determining the phoM map location in Escherichia coli K-12 by using a nearby transposon Tn10 insertion.

A phoR strain was constructed with transposon Tn10 inserted near the phoM+ locus. This was done without any prior knowledge of the phoM map location. Subsequently, we defined the phoM map position by screening tetracycline-sensitive (Tcs) derivatives for mutants which were both alkaline phosphatase negative (ther phoR phoM double mutant phenotype) and auxotrophic simultaneously. Some of these mutants were Thr-. Bacteriophage P1-mediated transductions were used to confirm that phoM and its nearby Tn10 insertion were closely linked to thr. Unexpectedly, 7 of 10 mutants analyzed also had mutations unlinked to the phoM-thr-Tn10 region. These may represent a new type of Tn10-promoted molecular event which is caused by transposition of a Tn10 end (IS10).

Alkaline Phosphatase↗
PubMed1981

Use of bacteriophage transposon Mu d1 to determine the orientation for three proC-linked phosphate-starvation-inducible (psi) genes in Escherichia coli K-12.

We have previously used the bacteriophage transposon Mu d1 (which encodes the lacZY structural genes but without their promoter) to construct strains that have lacZY fused to phosphate-regulated promoters in Escherichia coli K-12. Among 18 identified phosphate-starvation-inducible (psi) genes, three (the phoA and two new genes: psiF and psiG) are closely linked to the proC region. The gene order (clockwise) is phoA psiF proC psiG phoB phoR. Using these mutants containing Mu d1 insertions, we devised and tested a new method to determine their orientation. In this procedure, mutants with deletions that are selectable by their ability to grow at 42 degrees C are tested for the presence of Mu d1 and of neighboring genes. Some difficulties arose during analysis of suspected deletion-containing strains derived from Mu d1 lysogens (which also contained a Tn5 element) that were caused by Mu d1 and transposon transpositions and other possible genome rearrangements. Nevertheless, we have shown that the phoA and psiF genes are transcribed clockwise and the psiG gene is transcribed counterclockwise towards proC. Because phoA, but not psiF, gene expression requires the phoB+ (positive regulator) gene product, the phoA and psiF genes do not constitute an operon. On the other hand, the psiG:lacZ fusion-bearing strain may have a fusion to the promoter-distal end of the phoB gene. This implies that phoB expression is phosphate regulated. We believe that this method may be useful in general to elucidate the direction of gene transcription.

Chromosome Mapping↗