Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.     

Apolipoprotein composition of bovine lipoproteins isolated by gel filtration chromatography

1. Bovine lipoproteins were isolated from plasma by gel filtration and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. Bovine triglyceride-rich lipoproteins contained a novel low mol. wt protein Mr = 22,000 and low mol. wt proteins that may be analogous to non-ruminant apolipoproteins A-I, A-IV, and E. 3. Apolipoprotein C appeared to be a minor constituent of bovine triglyceride-rich lipoproteins. 4. Triglyceride-rich lipoproteins contained two high mol. wt proteins of approx. Mr = 220,000 and 290,000. 5. The predominant bovine low density lipoprotein apolipoprotein was approx. Mr = 290,000, however, greater then 25 proteins were often observed between Mr = 110,000 and 370,000. 6. Bovine high density lipoprotein contained proteins analogous to apolipoprotein A-I and C apolipoproteins. 7. Differences in apolipoprotein profiles between non-lactating and lactating cows were not apparent.     

Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum

We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited.     

Recombination sites in the HLA class II region are haplotype dependent

We have analyzed DNA sequence polymorphisms of DQ alpha and DQ beta chains from three haplotypes from the DRw52 family: DR5 DQw1 (FPA, GM3106), DRw6 DQw1 (CB6B, 10w9060), and DRw6 DQw3 (AMALA, 10w9064). The results indicate that the DR5 DQw1 and DRw6 DQw1 haplotypes have arisen by recombination between the DR beta 1 and DQ alpha loci. This contrasts with our previous analysis of DR4 DQ"Wa", DR3 DQ"Wa", and DR7 DQw3 haplotypes, all of which appear to have arisen by virtue of recombination between DQ alpha and DQ beta. Thus, there appear to be at least two different sites where recombination has occurred within the DR and DQ subregions. These differing patterns of recombination were interpreted in the context of the three major family groups of class II haplotypes, the DRw53, DRw52, and DR1/2 haplotype families. The data indicate that haplotypes from these family groups tend to undergo recombination at different locations. We propose that these differences in site of recombination are a reflection of differences in the molecular organization of the haplotypes belonging to each family group.     

When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs

Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.