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Phase variation of bacterial alkaline phosphatase
No full textThe pstS463 mutation, which was uncovered in a search for constitutive regulatory mutants of bacterial alkaline phosphatase (BAP) was found to cause BAP-phase variation. This phenotype is the alternation of enzyme synthesis between on and off states. This mutation is an IS2 insertion into the promoter of the pst operon which encodes the phosphate specific transport system. It reduces transcription through the operon. The pho regulatory genes are involved in regulating BAP-phase variation, as well as BAP expression in wild type cells. Mutations in phoB or phoR are epistatic to pstS463. Also diploidy for the phoBR region abolishes BAP-phase variation, producing BAP constitutive strains. Mutations in genes other than pho genes are also capable of abolishing BAP phase variation. Mutations in cya, crp and hupAB cause the pstS463 strains to exhibit a BAP repressed phenotype while mutations in himA or hip cause the pstS463 strains to exhibit a BAP constitutive phenotype. Possible mechanisms for these phenotypes are discussed
Finding new functions for the Ugp and PitA transport systems of Escherichia coli K-12
No full textEscherichia coli K-12 has two major systems for transporting inorganic phosphate (Pi). The high-affinity phosphate specific transport (Pst) system is an ABC transporter that is required for both P i uptake and Pi regulation of the Pho regulon. The low affinity Pi transporter (PitA) is a single transmembrane protein that does not belong to the Pho regulon. Besides, E. coli can utilize organic phosphate transported by several organic phosphate transporters which include Ugp system for transporting glycerol-6-phosphate. Here we discovered that under certain conditions PitA is required for proper assembly or functioning of the Pst transporter. Whereas a non-polar single-gene deletion of pstS (encoding the periplasmic P i binding protein) can be fully complemented by a single or multi-copy plasmid encoding PstS, a ΔpstS ΔpitA mutant cannot. Rather, a ΔpitA Δ pstS mutant can be restored to normal regulation only when genes for the entire pstSCAB region are expressed together from a single or multi-copy plasmid. These results provide the first evidence for a functional interaction between the PitA and PstSCAB transporters. By use of pitAp- and pitBp-lacZ transcriptional fusions, we show that these promoters are inducible under conditions of P i limitation in a PhoR/PhoB independent manner. Our data contradict the interpretations that PstS is not essential for Pi regulation of the Pho regulon (S. M. Hoffer, J. Tommassen, J. Bacteriol. 183: 5768-5783) and that PhoB negatively controls pitB expression (R. M. Harris, D. C. Webb, S. M. Howitt, G. B. Cox, J. Bacteriol. 183: 5008-5014). Unexpectedly, deleting genes for the PhoR/PhoB two-component system (TCS) resulted in synthetic lethality in a pstSCAB-phoU pitA pitB triple mutant due to an inability to assimilate inorganic or organic phosphate sources. Further experiments showed that growth of a pstSCAB-phoU pitA pitB mutant on organic or inorganic phosphates requires the Ugp system, whose expression is under PhoR/PhoB control. These data provide genetic evidence for uptake of Pi by the Ugp system. The finding that the Ugp system itself is capable of Pi uptake calls into question earlier evidence for control of Pho regulon gene expression, and the ugp operon in particular by internal phosphate
Functional studies on polar targeting and signaling of the PhoR protein in Escherichia coli
No full textA genome-wide study revealed that the PhoR histidine kinase is localized to the cell poles in Escherichia coli K-12 (WWW.ECOLIHUB.ORG/GENOBASE). Fluorescence imaging of a PhoR-Venus hybrid protein showed that the distribution of PhoR-Venus to the poles varies with the growth phase or cell cycle. During rapid growth, several patterns are observed. As cells enter the stationary phase, the fraction of cells showing polar localization dramatically increases. PhoR is inner membrane histidine kinase that is required for signal transduction in the control of the phosphate (Pho) regulon, whose expression is regulated by extracellular inorganic phosphate (Pi(ext) ). Similar patterns are seen during growth in Pi-limited and Pi excess conditions, thus suggesting that its distribution is unaffected by the Pi(ext) concentration. We further showed that the PhoR PAS domain (L107 to D193) is required for both polar localization and Pi signaling. Switching thirty-three residues lead to identifying four switches (T115A, TY153/155AA, R178A and R192A) that dramatically reduce the distribution of PhoR to the cell poles and two residues (D138 and R148) that are critical for signaling
Phosphate-independent controls of bacterial alkaline phosphatase synthesis in Escherichia coli K-12
No full textThe synthesis of bacterial alkaline phosphatase (Bap) is normally induced several hundredfold when phosphate is growth limiting. This control requires both products of the phoBR operon. PhoB is a transcriptional activator, and PhoR is a sensory protein that detects environmental phosphate levels. Earlier studies showed that in phoR mutants phoA transcription is no longer controlled by phosphate. Instead, its expression is regulated by the creABCD (formerly called phoM) operon. phoR mutants with a wild-type creABCD operon show induced synthesis of Bap on glucose medium, and an alternating pattern of Bap synthesis, termed Bap clonal variation, on TYE agar. Point mutations in the creC gene confer a Bap\sp- phenotype. However, deletion mutations can confer either a Bap\sp- or clonal variation phenotype, depending on whether neighboring genes are deleted and the strain background. Because strains with certain creABCD deletions are Bap variable, this suggested that gene products other than CreC may be involved in regulation of Bap synthesis. To determine which additional genes in the creABCD region are involved, this region was mutagenized with the transposon Tn5, and these insertions were used to construct new deletions. The results showed that (i) strains with non-polar insertions in the creA, creB, and creD genes show normal regulation of Bap synthesis; (ii) a strain with a deletion removing the creA and creB genes shows a Bap induced phenotype; (iii) strains with simple mutations in the creC gene are Bap\sp-; (iv) strains with insertions in the neighboring arcA gene are Bap induced; and (v) strains with deletions that remove both the creC and arcA genes are Bap\sp- on glucose and show the Bap clonal variation phenotype on TYE agar. Models are presented to explain how the products of the creABCD operon and the arcA gene regulate Bap synthesis. Several lines of evidence indicate that one or more additional controls regulate bap synthesis in a PhoR-and CreC-independent manner. As described above, arcA mutations restore Bap synthesis in phoR (creABCD) mutants. We found that an ompR mutation also restored Bap synthesis in phoR (creABCD) mutants. Furthermore, phoR (creABCD) mutants synthesize Bap when plated on TYEG agar. In order to study Pi-independent control of Bap synthesis, 74 transposon-induced mutants were isolated with altered Bap phenotypes. Thirty-two Bap\sp- mutants were shown to have insertions in the phoA or phoB genes. The remaining 42 mutants were placed into one of 19 mutant classes based on their Bap phenotypes or linkage data. Many insertions resulted in the induction of a phoR- and CreC-independent control of Bap synthesis, whereas some mutants eliminated the phoR- and CreC-independent Bap synthesis on TYEG agar. Representative insertions from several mutant classes were cloned and the DNA sequence of their fusion junctions were determined. Based on sequence and/or linkage data, the sites of the insertions for 11 mutants classes were identified. Interestingly, 4 mutants that conferred a Bap induced phenotype had insertions in the ackA gene, for acetate kinase. Based on subsequent experiments with these mutants, we conclude that one phoR- and CreC-independent control of Bap synthesis responds to acetyl phosphate or a closely related metabolite. Several other mutations that alter Bap synthesis in a phoR (creABCD) mutant may affect Bap synthesis indirectly by altering the level of acetyl phosphate
The identification of phosphate starvation-inducible genes and the role of the phosphate-specific transport (Pst) system andphoU genes in phosphate regulation in Escherichia coli
No full textAll living cells require phosphorus for growth. The means by which the enteric bacterium Escherichia coli satisfies this requirement and the coordinate regulation of the expression of genes involved in the utilization of various phosphorus sources is a model for cellular response to nutrient deprivation. When cells are limited for inorganic phosphate (Pi), the preferred phosphorus source, the transcription of the PHO regulon genes is markedly induced. This expression is dependent upon the PhoB and PhoR proteins which comprise a two-component regulatory system. The products of the pstSCAB-phoU operon are also involved in this regulation because mutations in these genes render the PHO regulon constitutive. The pst genes encode the high-affinity phosphate-specific transport system (Pst) while PhoU is thought to be a regulatory protein because a missense allele of phoU, phoU35, has no effect on transport by Pst. There are two interesting problems regarding the PHO regulon and its control that have been addressed in this work. First, previous studies have indicated that more proteins have higher levels of synthesis during Pi starvation than have been thus far identified. Through the analysis of a set of random lacZ fusions to unknown promoters (generated by Mu d1) isolated for phosphate starvation-inducible (psi) Lac expression, unknown Pi-regulated genes were studied. Several psi genes were identified by DNA sequence analysis of the psi::lacZ(Mu d1) fusion junctions and novel regulatory interactions have been explored by the study of the regulation of psi::lacZ fusions. The mechanisms of control of the PHO regulon have also been investigated. Most of the work implicating the genes of the pstSCAB-phoU operon in Pi repression of the PHO regulon involves the analysis of mutations, including phoU35, isolated under conditions that should not have permitted Pst\sp- cells to grow. Defined deletions of the pstSCAB-phoU operon have been constructed, verified, and studied. These analyses also indicate that cells which lack PhoU but retain a functional Pst system have a severe growth defect. Additionally, when these phoU null mutants are starved for Pi they become sensitive to Pi and can only recover from these starvation conditions when provided with an alternate phosphorus source in the absence of Pi. Thus, PhoU is a bifunctional protein involved in Pi utilization and in PHO regulon control
Development and use of new molecular and genomic tools for studying Escherichia coli and other bacteria
No full textGenetic tools for constructing gene disruptions and reporter fusions play an important role in gene function and regulation studies for all organisms, no matter the subject is a eubacterium, such as E. coli, or a higher eukaryotic organism, such as a mouse. These studies have contributed significantly to our current knowledge in life sciences, such as bacterial virulence, cell division, cell development, and human diseases, etc. To facilitate functional genomic studies in E. coli and other bacteria, a new genetic tool for high-throughput (HT) studies, a conjugative att shuttle (CAS) CRIM system, has been developed in this work, and has been shown to work in diverse Gram-negative bacteria, including Pseudomonas aeruginosa, Shewanella oneidensis, and Vibrio cholerae. Signal transduction by protein phosphorylation plays a central role in regulation of numerous cellular processes. Two component systems (TCSs) are the most prevalent signal transduction systems in bacteria. With a goal towards understanding the roles of all E. coli TCSs and regulatory interactions among them, we have constructed defined deletion mutants for each TCS systematically and then analyzed these mutants with a new technology called Phenotype MicroArrays that permits assaying nearly 2000 growth phenotypes simultaneously. New roles for some TCSs were uncovered and potential regulatory interactions among different TCSs are discussed. In particular, the PhoR(HK)/PhoB(RR) TCS, which controls genes for phosphorus acquisition, has been investigated in several aspects. Eleven putative PhoR/PhoB regulated promoters were tested for their expression dependency on the PhoR/PhoB TCS under phosphate (Pi) starvation. Roles of the PboR/PhoB TCS in transcription of several polyphosphate kinase (PPK) orthologs under Pi starvation were also examined by using the CAS CRIM system. Results showed that there are two groups of PPK orthologs, one is PhoR/PhoB dependent and the other is PhoR/PhoB independent. Five additional noncognate HKs besides CreC were shown to activate PhoB by earlier bulk enzyme assays. Single cell studies of cross activation of PhoB by these six HKs were carried out in this work and stochastic characteristics of cross talk among TCSs were uncovered and are discussed
Two types of transcriptional controls on polyphosphate kinase (PPK) gene promoters in different bacteria
No full textIn bacteria, polyphosphate kinase (PPK) is responsible for the synthesis of polyP from ATP and the reversible conversion of polyP and ADP to ATP. PPK plays a crucial role in the ability of bacteria to adapt to nutritional stringencies and environmental stresses. Bacterial ppk gene expression was tested at the transcriptional level by using a ppk-promoter lacZ fusion reporter system under a variety of stressful conditions. The expression of ppk gene was greatly induced (7-20 fold higher depending on stress or medium) by deficiencies of phosphate, amino acids, or during entry into stationary phase. In this work, evidence was found for two different modes of regulation of ppk transcription in bacteria. In E. coli and S. enterica, ppk expression in response to starvation appears to be dependent on the ppGpp/DksA system and independent of the PhoR/PhoB two-component system under conditions of phosphate starvation. In contrast, increased ppk expression in response to phosphate starvation is dependent on the PhoR/PhoB two-component system in Acinetobacter. sp. ADP1, K. pneumoniae, and V. cholerae. The activity of the ppk promoter is also greatly induced by purine starvation. Purine deficiency seems to be a common stimulus for the induction of ppk expression in both of the above categories of bacteria. However, the induction of ppk transcription by purine starvation seems to be independent of both the ppGpp/DksA system and the PhoR/PhoB two-component system. This result implies the presence of third control system in ppk expression
Molecular genetic analysis of the Escherichia coli phn(psiD) gene cluster and its role in phosphonate metabolism
No full textThe gram-negative bacterium Escherichia coli can use phosphonates (Pn), a class of compounds containing extremely stable carbon-phosphorus (C-P) bonds, as a sole phosphorus (P) source. Pn are metabolized in E. coli by the enzyme C-P lyase, which is poorly understood due to the inability to detect its activity in vitro. This difficulty has been overcome using a molecular genetic approach in the study of Pn metabolism. Earlier studies showed that Pn degradation required the phn gene cluster which, based on its DNA sequence, was proposed to contain seventeen genes, phnA through phnQ. However, the functions of individual genes in Pn utilization were undefined. In this study a detailed analysis of the phn gene cluster is presented. Phenotypic analysis of phn mutants revealed three distinct functions for the phn locus. These are: (i) C-P lyase activity, (ii) phosphite oxidation, a previously unsuspected but related activity, and (iii) the nonspecific transport of phosphorus compounds including Pn, phosphite, phosphate and phosphate esters. Using molecular genetic techniques the roles for each of the seventeen phn genes were defined. First, three genes (phnA, phnB and phnQ) were shown to have no role in Pn metabolism because plasmid subclones lacking these genes can still complement phn deletion mutants. The remaining fourteen genes were assigned a role(s) based on mutant analysis. A total of 53 independent transposon-induced mutants were characterized; these include 3 Mu d1, 7 Tn5 and 43 TnphoA\sp\prime prime insertions. The mutations were localized to an individual phn gene by DNA sequencing of the transposon insertion site. All fourteen genes were mutated at least once. Both polar and non-polar insertion mutants were studied at 20 sites. Each TnphoA\sp\prime prime insertion was characterized with respect to transcription. Eight insertions were also characterized with respect to translation and protein localization after conversion of the insertion to a lacZ or phoA protein fusion. The results showed: (i) three genes (phnC, phnD, and phnE) comprise a binding-protein dependent Pn transport system; (ii) eight genes (phnG, phnH, phnI, phnJ, phnK, phnL, phnM and phnP) are required for the utilization of both Pn and phosphite, suggesting that their products form a multi-subunit enzyme complex needed for both activities; (iii) one gene (phnN) is not absolutely required for catalysis, although it seems to modulate enzyme activity; and (iv) two genes (phnF and phnO) have no biochemical functions and, based on protein homologies, are probably involved in gene regulation. Lastly, the regulation of the phn gene cluster was examined. The phn gene cluster was shown to comprise an operon of 10.9 kb, which is the largest yet reported in E. coli. An internal site for down-regulation of phn operon transcription was also revealed, which lies between the phnE and phnF genes
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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