Joe A. Fralick

Phage Therapy of Pseudomonas aeruginosa Infection in a Mouse Burn Wound Model

ABSTRACT Mice compromised by a burn wound injury and subjected to a fatal infection with Pseudomonas aeruginosa were administered a single dose of a Pseudomonas aeruginosa phage cocktail consisting of three different P. aeruginosa phages by three different routes: the intramuscular (i.m.), subcutaneous (s.c.), or intraperitoneal (i.p.) route. The results of these studies indicated that a single dose of the P. aeruginosa phage cocktail could significantly decrease the mortality of thermally injured, P. aeruginosa-infected mice (from 6% survival without treatment to 22 to 87% survival with treatment) and that the route of administration was particularly important to the efficacy of the treatment, with the i.p. route providing the most significant (87%) protection. The pharmacokinetics of phage delivery to the blood, spleen, and liver suggested that the phages administered by the i.p. route were delivered at a higher dose, were delivered earlier, and were delivered for a more sustained period of time than the phages administered by the i.m. or s.c. route, which may explain the differences in the efficacies of these three different routes of administration.

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae

Multidrug‐resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V. cholerae, we isolated a DNA fragment (pVC) that enabled an E. coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E. coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V. cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V. cholerae. In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.

Genomic Organization and Molecular Characterization of Clostridium difficile Bacteriophage ΦCD119

In this study, we have isolated a temperate phage (ΦCD119) from a pathogenic Clostridium difficile strain and sequenced and annotated its genome. This virus has an icosahedral capsid and a contractile tail covered by a sheath and contains a double-stranded DNA genome. It belongs to the Myoviridae family of the tailed phages and the order Caudovirales. The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. The DNA sequence of this phage consists of 53,325 bp, which carries 79 putative open reading frames (ORFs). A function could be assigned to 23 putative gene products, based upon bioinformatic analyses. The ΦCD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The ΦCD119 attachment site attP lies in a noncoding region close to the putative integrase (int) gene. We have identified the phage integration site on the C. difficile chromosome (attB) located in a noncoding region just upstream of gene gltP, which encodes a carrier protein for glutamate and aspartate. This genetic analysis represents the first complete DNA sequence and annotation of a C. difficile phage.

Erwinia chrysanthemi tolC Is Involved in Resistance to Antimicrobial Plant Chemicals and Is Essential for Phytopathogenesis

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.

A strand-specific model for chromosome segregation in bacteria

Chromosome separation and segregation must be executed within a bacterial cell in which the membrane and cytoplasm are highly structured. Here, we develop a strand‐specific model based on each of the future daughter chromosomes being associated with a different set of structures or hyperstructures in an asymmetric cell. The essence of the segregation mechanism is that the genes on the same strand in the parental cell that are expressed together in a hyperstructure continue to be expressed together and segregate together in the daughter cell. The model therefore requires an asymmetric distribution of classes of genes and of binding sites and other structures on the strands of the parental chromosome. We show that the model is consistent with the asymmetric distribution of highly expressed genes and of stress response genes in Escherichia coli and Bacillus subtilis. The model offers a framework for interpreting data from genomics.

A SeqA hyperstructure and its interactions direct the replication and sequestration of DNA

A level of explanation in biology intermediate between macromolecules and cells has recently been proposed. This level is that of hyperstructures. One class of hyperstructures comprises the genes, mRNA, proteins and lipids that assemble to fulfil a particular function and disassemble when no longer required. To reason in terms of hyperstructures, it is essential to understand the factors responsible for their formation. These include the local concentration of sites on DNA and their cognate DNA‐binding proteins. In Escherichia coli, the formation of a SeqA hyperstructure via the phenomenon of local concentration may explain how the binding of SeqA to hemimethylated GATC sequences leads to the sequestration of newly replicated origins of replication.

Isolation and Characterization of VceC Gain-of-Function Mutants That Can Function with the AcrAB Multiple-Drug-Resistant Efflux Pump of Escherichia coli

VceC is the outer membrane component of the major facilitator (MF) VceAB-VceC multiple-drug-resistant (MDR) efflux pump of Vibrio cholerae. TolC is the outer membrane component of the resistance-nodulation-division AcrAB-TolC efflux pump of Escherichia coli. Although these proteins share little amino acid sequence identity, their crystal structures can be readily superimposed upon one another. In this study, we have asked if TolC and VceC are interchangeable for the functioning of the AcrAB and VceAB pumps. We have found that TolC can replace VceC to form a functional VceAB-TolC MDR pump, but VceC cannot replace TolC to form a functional AcrAB-VceC pump. However, we have been able to isolate gain-of-function (gof) VceC mutants which can functionally interface with AcrAB. These mutations map to four different amino acids located at the periplasmic tip of VceC. Chemical cross-linkage experiments indicate that both wild-type and gof mutant VceC can physically interact with the AcrAB complex, suggesting that these gof mutations are not affecting the recruitment of VceC to the AcrAB complex but rather its ability to functionally interface with the AcrAB pump.

Characterization of the Vibrio cholerae vceCAB Multiple-Drug Resistance Efflux Operon in Escherichia coli

Herein, we identify vceC as a component of a vceCAB operon, which codes for the Vibrio cholerae VceAB multiple-drug resistance (MDR) efflux pump, and vceR, which codes for a transcriptional autoregulatory protein that negatively regulates the expression of the vceCAB operon and is modulated by some of the substrates of this MDR efflux pump.

Evidence that Clostridium difficile TcdC Is a Membrane-Associated Protein

Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile.

Hyperstructures, genome analysis and I-Cells

New concepts may prove necessary to profit from the avalanche of sequence data on the genome, transcriptome, proteome and interactome and to relate this information to cell physiology. Here, we focus on the concept of large activity-based structures, or hyperstructures, in which a variety of types of molecules are brought together to perform a function. We review the evidence for the existence of hyperstructures responsible for the initiation of DNA replication, the sequestration of newly replicated origins of replication, cell division and for metabolism. The processes responsible for hyperstructure formation include changes in enzyme affinities due to metabolite-induction, lipid-protein affinities, elevated local concentrations of proteins and their binding sites on DNA and RNA, and transertion. Experimental techniques exist that can be used to study hyperstructures and we review some of the ones less familiar to biologists. Finally, we speculate on how a variety of in silico approaches involving cellular automata and multi-agent systems could be combined to develop new concepts in the form of an Integrated cell (I-cell) which would undergo selection for growth and survival in a world of artificial microbiology.