Sankar Adhya

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Papers by Sankar Adhya

Phosphoglucomutase Mutants of <i>Escherichia coli</i> K-12

Journal of Bacteriology, Nov 1, 1971

Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mut... more Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accu- mulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-l-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glu- cose-l-phosphate is also strongly depressed, since amino acid-or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm muta- tions isolated map at about 16 min on the genetic map, between purE and the gal operon.

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GalR Mediated Interactions Across the E. Coli Chromosome

Biophysical Journal, 2012

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Inactivation of Metabolic Genes Causes Short- and Long-Range dys-Regulation in Escherichia coli Metabolic Network

PLOS ONE, Dec 5, 2013

The metabolic network in E. coli can be severely affected by the inactivation of metabolic genes ... more The metabolic network in E. coli can be severely affected by the inactivation of metabolic genes that are required to catabolize a nutrient (D-galactose). We hypothesized that the resulting accumulation of small molecules can yield local as well as systemic effects on the metabolic network. Analysis of metabolomics data in wild-type and Dgalactose non-utilizing mutants, galT, galU and galE, reveal the large metabolic differences between the wild-type and the mutants when the strains were grown in D-galactose. Network mapping suggested that the enzymatic defects affected the metabolic modules located both at short-and long-ranges from the D-galactose metabolic module. These modules suggested alterations in glutathione, energy, nucleotide and lipid metabolism and disturbed carbon to nitrogen ratio in mutant strains. The altered modules are required for normal cell growth for the wild-type strain, explaining why the cell growth is inhibited in the mutants in the presence of D-galactose. Identification of these distance-based dys-regulations would enhance the systems level understanding of metabolic networks of microorganisms having importance in biomedical and biotechnological research.

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Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation

Proceedings of the National Academy of Sciences of the United States of America, Oct 15, 1992

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requir... more The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMPinduced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMPindependent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D a-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D a-helices to come together. As a consequence, the F a-helix is released from the D a-helix and can interact with DNA. At position 144 in the D a-helix and within interacting distances of the F a-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F a-helix may be a way of making the helix available for DNA binding. We believe that the cAMP- induced allosteric change involves similar hinge reorientation to adjust the C and D a-helices, allowing outward movement of the F a-helix.

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λ Recombineering Used to Engineer the Genome of Phage T7

Antibiotics, 2020

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology t... more Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

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Nucleoid Remodeling During Environmental Adaptation as Seen Through Soft X-ray Tomography

Microscopy and Microanalysis, 2020

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Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase

Journal of Bacteriology, 1981

Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has be... more Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase.

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Glucose Effect and the Galactose Enzymes of Escherichia coli : Correlation Between Glucose Inhibition of Induction and Inducer Transport

Journal of Bacteriology, 1966

Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols . Glucose effect and the ga... more Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols . Glucose effect and the galactose enzymes of Escherichia coli : correlation between glucose inhibition of induction and inducer transport. J. Bacteriol. 92: 601–608. 1966.—The inhibitory effect of glucose on the induction of the enzymes required for galactose utilization (“glucose effect”) was studied in Escherichia coli . Experiments on the uptake into the cell of labeled inducers ( d -galactose- C 14 and d -fucose- H 3 ) pointed to inhibition at the level of inducer transport as the possible primary mechanism of the glucose effect in the case of the gal enzymes. This interpretation was supported by the finding that a mutant constitutive for the lac enzymes was resistant to glucose inhibition of galactose induction of the gal enzymes; the mutant had acquired a glucose-resistant alternative transport mechanism for galactose via the constitutively synthesized galactoside permease. Further support for the transport...

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Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption

mBio, 2020

The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of diff... more The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo , then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

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Processing generates 3′ ends of RNA masking transcription termination events in prokaryotes

Proceedings of the National Academy of Sciences, 2019

Significance Transcription termination by RNA polymerase in prokaryotes is well understood in con... more Significance Transcription termination by RNA polymerase in prokaryotes is well understood in contrast to similar mechanisms in higher organisms. Despite the in vitro occurrence of two types of demonstrable transcription termination events in prokaryotes at the end of transcription units, they are obscured in vivo in two ways: suppression of termination by traversing of the RNA polymerase through the termination sites when coupled to translation, or by further processing of the actual terminated RNA 3′ ends by RNases, as in eukaryotes.

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Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation

mBio, Jan 17, 2017

Bacteriophages infect an estimated 10(23) to 10(25) bacterial cells each second, many of which ca... more Bacteriophages infect an estimated 10(23) to 10(25) bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased commun...

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Ribosomal protein S1 promotes transcriptional cycling

RNA, 2006

Prokaryotic RNA polymerases are capable of efficient, continuous synthesis of RNA in vivo, yet pu... more Prokaryotic RNA polymerases are capable of efficient, continuous synthesis of RNA in vivo, yet purified polymerase-DNA model systems for RNA synthesis typically produce only a limited number of catalytic turnovers. Here, we report that the ribosomal protein S1—which plays critical roles in translation initiation and elongation in Escherichia coli and is believed to stabilize mRNA on the ribosome—is a potent activator of transcriptional cycling in vitro. Deletion of the two C-terminal RNA-binding modules—out of a total of six loosely homologous RNA-binding modules present in S1—resulted in a near-loss of the ability of S1 to enhance transcription, whereas disruption of the very last C-terminal RNA-binding module had only a mild effect. We propose that, in vivo, cooperative interaction of multiple RNA-binding modules in S1 may enhance the transcript release from RNA polymerase, alleviating its inhibitory effect and enabling the core enzyme for continuous reinitiation of transcription.

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Toward a live microbial microbicide for HIV: Commensal bacteria secreting an HIV fusion inhibitor peptide

Proceedings of the National Academy of Sciences, 2005

Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal t... more Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli , secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the...

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Complementation, Recombination, and Suppression in Galactose Negative Mutants of E. Coli

Proceedings of the National Academy of Sciences, 1963

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Spiral structure of Escherichia coli HUαβ provides foundation for DNA supercoiling

Proceedings of the National Academy of Sciences, 2007

We determined the crystal structure of the Escherichia coli nucleoid-associated HUαβ protein by x... more We determined the crystal structure of the Escherichia coli nucleoid-associated HUαβ protein by x-ray diffraction and observed that the heterodimers form multimers with octameric units in three potential arrangements, which may serve specialized roles in different DNA transaction reactions. It is of special importance that one of the structures forms spiral filaments with left-handed rotations. A negatively superhelical DNA can be modeled to wrap around this left-handed HUαβ multimer. Whereas the wild-type HU generated negative DNA supercoiling in vitro , an engineered heterodimer with an altered amino acid residue critical for the formation of the left-handed spiral protein in the crystal was defective in the process, thus providing the structural explanation for the classical property of HU to restrain negative supercoils in DNA.

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Corrections: An Arcane Role of DNA in Transcription Activation

Proceedings of the National Academy of Sciences, 1994

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Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target

Proceedings of the National Academy of Sciences, 1996

Genetic instability is thought to be responsible for the numerous genotypic changes that occur du... more Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lac...

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Role of domain II of Pseudomonas exotoxin in the secretion of proteins into the periplasm and medium by Escherichia coli

Proceedings of the National Academy of Sciences, 1988

Pseudomonas exotoxin (PE) is composed of structural domains I, II, and III; when interacting with... more Pseudomonas exotoxin (PE) is composed of structural domains I, II, and III; when interacting with mammalian cells the function of domain I is cell recognition, the function of domain II is membrane translocation, and domain III functions in ADP ribosylation. PE is secreted by Pseudomonas aeruginosa into its growth medium. The domain responsible for secretion has been examined by expressing modified PE genes in Escherichia coli under the control of a T7 promoter. Without a signal sequence, PE accumulates within the cell, but PE is secreted into the periplasm when part or all of domain I is removed. PE appears in the periplasm and medium when domain I and part of domain II are removed. Domain II alone is secreted into the periplasm, whereas domain III alone remains within the cell. Addition of an OmpA signal sequence results in secretion of mature PE into the periplasm and secretion of domains II-III into the medium. A protein composed of transforming growth factor alpha fused to the ...