Sankar Adhya

Functional domains of pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli

Pseudomonas exotoxin A is a single chain toxin with three structural domains that inhibits protein synthesis in eukaryotic cells by catalyzing ADP ribosylation of elongation factor 2. To study the function of these domains, we deleted different portions of the PE structural gene and expressed these constructs in E. coli using an inducible T7 promoter. These studies indicate that structural domain Ia is required for cell recognition, that structural domain II is required to translocate the toxin across a cellular membrane, and that structural domain III and a portion of domain Ib are required for ADP ribosylation activity. Toxin lacking domain Ia is about 100-fold less toxic to mice than intact PE and should be a useful molecule for the construction of immunotoxins.

Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium

ABSTRACT Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 109 CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 × 108 PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.

Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector.

The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment [Wu and Crothers, Nature 308 (1984) 509-513]. We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations. The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites. The protein-binding sequence is inserted at one of these cloning sites. To investigate the functional significance of bending, we have compared, using pBend2, the cAMP.cAMP-receptor protein (CPR)-induced bending of CRP-binding sites found in five different genes of Escherichia coli. We have also shown that the bacteriophage lambda 0R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.

The prospect for bacteriophage therapy in Western medicine

Bacteriophage (phage) have been used for clinical applications since their initial discovery at the beginning of the twentieth century. However, they have never been subjected to the scrutiny — in terms of the determination of efficacy and pharmacokinetics of therapeutic agents — that is required in countries that enforce certification for marketed pharmaceuticals. There are a number of historical reasons for this deficiency, including the overshadowing discovery of the antibiotics. Nevertheless, present efforts to develop phage into reliable antibacterial agents have been substantially enhanced by knowledge gained concerning the genetics and physiology of phage in molecular detail during the past 50 years. Such efforts will be of importance given the emergence of antibiotic-resistant bacteria.

Repressor induced site-specific binding of HU for transcriptional regulation

Transcription from two overlapping gal promoters is repressed by Gal repressor binding to bipartite gal operators, OE and OI, which flank the promoters. Concurrent repression of the gal promoters also requires the bacterial histone‐like protein HU which acts as a co‐factor. Footprinting experiments using iron–EDTA‐coupled HU show that HU binding to gal DNA is orientation specific and is specifically dependent upon binding of GalR to both OE and OI. We propose that HU, in concert with GalR, forms a specific nucleoprotein higher order complex containing a DNA loop. This way, HU deforms the promoter to make the latter inactive for transcription initiation while remaining sensitive to inducer. The example of gal repression provides a model for studying how a ‘condensed’ DNA becomes available for transcription.

Transcription antitermination by bacteriophage lambda N gene product

Abstract In the presence of the lambda N gene product, transcription originating at the prophage p L promoter can extend beyond termination signals and into the neighboring bacterial gal operon. In Escherichia coli rho mutants, N function is still required for p L transcription to enter gal . The requirement for N function in rho mutants is abolished by deletion of the prophage genome between 57.4 and 70.9 lambda fractional lengths. These results suggest: 1. (1) Rho-independent termination sites lie in the p L operon of lambda. 2. (2) Both Rho-independent and Rho-dependent terminators are suppressed by N function. N product must, therefore, be considered as a transcription antitermination factor, rather than as an antagonist of Rho, as previously suggested. Various DNA segments have been interposed between p L and gal and their effect on gal escape synthesis studied. Lambda DNA encoding the 6 S transcript, which ends at a Rho-independent terminator, does not reduce gal escape synthesis. The J-b2 region of the phage chromosome, however, completely blocks p L -promoted gal enzyme synthesis; the block is partially relieved by the rho15 mutation. The introduction of large DNA segments lowers gal escape in both rho + and rho15 mutants. The extent of gal escape is directly proportional to the efficiency of suppression of a prophage Nam mutation, suggesting that N product is not made in excess.

Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters

Abstract Our results demonstrate the existence of two initiation sites, S 1 and S 2 , for transcription of the galactose operon of E. coli. Transcription from each of these sites responds to different regulatory mechanisms. In the presence of Cyclic AMP Receptor Protein (CRP) and cyclic AMP (cAMP), transcription initiates only at S 1 . In the absence of CRP or cAMP, transcription starts from S 2 . Examination of a gal promoter mutation shows that transcription from S 1 is abolished, but that initiation can occur at S 2 and is still subject to repression by CRP-cAMP. In vivo gal expression from this mutant promoter is actually increased when cyclic AMP or CRP are eliminated by mutations in the cya (adenylate cyclase) or crp genes. Studies of gal expression from a wild-type promoter in cya and crp mutants are also consistent with the use of the two startpoints in vivo and suggest that when CRP or cAMP is absent, transcription starts from S 2 in vivo. S 1 corresponds to the startpoint for CRP- and cAMP-dependent gal mRNA previously determined (Musso et al., 1977). S 2 precedes S 1 by 5 base pairs in the DNA of the gal regulatory region. A heptamer sequence analogous to those described for other promoters precedes each startsite by a distance of 6 base pairs. DNA sequence analyses of the gal promoter mutation mentioned above establish that the base change is in the second residue of the heptamer preceding S 1 .

Bacteriophage K1-5 Encodes Two Different Tail Fiber Proteins, Allowing It To Infect and Replicate on both K1 and K5 Strains of Escherichia coli

ABSTRACT A virulent double-stranded DNA bacteriophage, ΦK1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of thePodoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of ΦK5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of ΦK1E, which allows this phage to specifically infect K1 strains ofE. coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of ΦK5, ΦK1E, and ΦK1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.

A control element within a structural gene: The gal operon of Escherichia coli

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.

The galactose regulon of Escherichia coli

Galactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by two different galactose‐specific transport systems. At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences. These sequences are recognized by at least three regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The negative regulators, GalR and GalS, discriminate between utilization of the high‐affinity (regulated by GalS) and low‐affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by CRP. Both promoters are strongly repressed by GalR but weakly by GalS. All but one of the constituent promoters of the gal regulon have two operators. The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.