Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

Characterisation of the major porins OmpU and OmpT of Vibrio cholerae

PhD ThesisThe asymmetric outer membrane (OM) of a Gram-negative bacterium has many proteins embedded as β-barrel structures in it called outer membrane proteins (OMPs). The majority of these OMPs (porins) form non-selective channels across the OM to allow passive uptake of substrates. The treatment for infections caused by such bacteria mostly involves the administration of drugs/antibiotics, for which these porins play a very crucial role by providing an efficient (although not yet fully understood) route through their channel. The goal of this study is to study small-molecule permeation through the major porins, OmpU and OmpT, of Vibrio cholerae (the causative agent of cholera) for potential use of these proteins as the target for designing antibiotics or vaccines. Towards this project, we have succeeded in solving the 3D X-ray crystal structures of OmpU and OmpT as well as the structures of the major porins from Klebsiella pneumoniae (OmpK36) and Enterobacter cloacae (OmpE36, OmpE35). The proteins (OmpU/T, OmpE35/E36 and OmpK36) show the typical arrangement of porins with three β-barrel monomers arranged into a trimer. Each monomer displays 16 antiparallel β-strands forming the hollow β-barrel formed by 8 long extracellular loops and 8 short periplasmic turns. The latching loop L2 stabilises the trimer while loop L3 departs from the β-barrel fold and constricts the pore half-way through the channel. An unusual feature is observed in the channels of OmpU and OmpT that distinguishes them from other typical porins. In OmpU, the first 10 residues of N-terminus insert into the barrel and constrict the pore. In contrast, the structure of OmpT reveals that the extracellular loop L8 folds inwards to constrict the lumen of the channel. Such constriction elements not only reduce the pore sizes of OmpU and OmpT but may also dramatically affect the internal electrostatics of these channels, which is very important for small-molecule permeation. In addition, we also performed single channel electrophysiology experiments with OmpU and OmpT which revealed interesting features with the addition of carbapenems.European Union’s Seventh Framework Programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in kind contribution. Therefore, a very special gratitude goes out to all down to EU Marie Curie network (ITN) for funding my PhD

TIM-Finder: A new method for identifying TIM-barrel proteins

<p>Abstract</p> <p>Background</p> <p>The triosephosphate isomerase (TIM)-barrel fold occurs frequently in the proteomes of different organisms, and the known TIM-barrel proteins have been found to play diverse functional roles. To accelerate the exploration of the sequence-structure protein landscape in the TIM-barrel fold, a computational tool that allows sensitive detection of TIM-barrel proteins is required.</p> <p>Results</p> <p>To develop a new TIM-barrel protein identification method in this work, we consider three descriptors: a sequence-alignment-based descriptor using PSI-BLAST e-values and bit scores, a descriptor based on secondary structure element alignment (SSEA), and a descriptor based on the occurrence of PROSITE functional motifs. With the assistance of Support Vector Machine (SVM), the three descriptors were combined to obtain a new method with improved performance, which we call TIM-Finder. When tested on the whole proteome of <it>Bacillus subtilis</it>, TIM-Finder is able to detect 194 TIM-barrel proteins at a 99% confidence level, outperforming the PSI-BLAST search as well as one existing fold recognition method.</p> <p>Conclusions</p> <p>TIM-Finder can serve as a competitive tool for proteome-wide TIM-barrel protein identification. The TIM-Finder web server is freely accessible at <url>http://202.112.170.199/TIM-Finder/</url>.</p

Molecular evolution of outer membrane proteins and characterization of temperate bacteriophages of Pasteurella multocida strains from different host species

Pasteurella multocida is a Gram-negative commensal bacterium which resides in the upper respiratory tract of mammals and birds. The organism is responsible for a variety of economically important diseases in a wide range of domestic animal species. It causes fowl cholera of poultry, haemorrhagic septicaemia of cattle and water buffalo, atrophic rhinitis of pigs, and pneumonia of cattle, sheep and pigs. In the present study, P. multocida isolates were selected based on an established framework of evolutionary relationships among 123 isolates of P. multocida based on the concatenated partial sequences (3990 bp) of seven housekeeping enzyme genes (Davies et al., unpublished; http://pubmlst.org/pmultocida_multihost). The isolates were recovered from different host species (cattle, sheep, pigs and poultry) and were associated with different diseases. The isolates represented various capsular serotypes, outer membrane protein (OMP)-types, 16S rRNA types, and sequence types. Phylogenetic trees based on the concatenated partial sequences (3990 bp) of the seven housekeeping enzyme genes, complete sequences (22,371 bp) of fifteen housekeeping enzyme genes and the core genome were almost identical in their topographies. The trees represented eight major groups or clusters of isolates and these clusters could also be defined, to varying degrees, by the host species of origin and/or disease syndrome. The pattern of clustering of isolates associated with different host species also demonstrated that transmission of P. multocida has occurred between different host species. Such host-switching could play an important role in generating diversity within P. multocida. Comparative nucleotide sequence analysis of genes encoding the predicted outer membrane proteins of different functions was carried out in 40 isolates of P. multocida to investigate nucleotide diversity and to assess the roles horizontal DNA transfer and recombination in the evolution and diversification of P. multocida. Comparative nucleotide sequence analysis provided clear evidence that horizontal DNA transfer and recombination (both intragenic and assortative) have occurred within the genes encoding P. multocida OMPs. However, it was also demonstrated that this varied from gene-to-gene. Four functional groups of OMPs were predicted based on the prediction analyses and these functions include outer membrane biogenesis and integrity (12 VIII proteins), transport and receptor (25 proteins), adherence (7 proteins) and enzymatic activity (9 proteins). Thirty five OMPs were analysed in this study in detail. The results showed limited levels of nucleotide and amino acid sequence variation was found within the genes encoding selected proteins with the exception of OmpA and OmpH1. However, there was evidence of gene exchange (assortative recombination) between isolates from different host species and divergent genetic lineages. High levels of nucleotide and amino acid sequence variation was found within two major surface-exposed proteins, OmpA and OmpH1. The results indicated that the ompA and ompH1 genes have undergone extensive horizontal DNA transfer, intragenic and assortative recombination. Variation in OmpA and OmpH1 occurred predominantly in the surface-exposed loop regions. There was strong evidence that natural selection is driving diversification of the hypervariable extracellular loop regions in both proteins. The diversity and molecular evolutionary relationships of ompA were further investigated in a larger selection of 74 P. multocida isolates. Sequence analysis of the 26 different ompA-type alleles revealed that the P. multocida ompA gene has undergone multiple horizontal gene transfer and recombination events because complex mosaic structures were found between ompA alleles. The diversity of temperate bacteriophages was examined in 47 P. multocida isolates. Phage particles were induced with mitomycin C and characterised morphologically by transmission electron microscopy (TEM). The phage particles were morphologically diverse and represented both the Siphoviridae and Myoviridae families. Both Siphoviridae and Myoviridae phage types were induced in certain isolates indicating that a single host may harbour multiple prophages. Moreover, phage DNA was successfully isolated from 18 P. multocida isolates. Bacteriophage DNA from isolates PM86, PM172, PM486, PM934 and PM954 showed the presence of two bands of different molecular size. Although these phages had a distinct Myoviridae-type morphology, they possessed an unusually small capsid as identified by TEM. Taken together, these results suggest the presence of phage-inducible chromosomal islands (PICIs), in these P. multocida strains. To date, PICIs have not been described in P. multocida. Genetic diversity of temperate bacteriophages was assessed by restriction endonuclease (RE) analysis and 10 different RE types (A to J) were identified. Plaque assay appeared to be IX less sensitive than TEM for detection of temperate bacteriophages. Only 11 (38%) P. multocida phages produced signs of infection against indicator strains. Nucleotide sequence analysis of phage genomic DNA from the same isolates demonstrated that both λ-like and Mu-like phages are induced in the same isolates of P. multocida. The results also showed that more than one λ-like and Mu-like phage is induced in the majority of isolates. Annotation of the sequenced phages resulted in five different Mu-like phages, one phage-inducible chromosomal island and seven λ-like phages. Further bacterial genome analyses identified additional intact prophages within the genomes of 40 isolates. From one to five intact prophages and prophage-like elements were identified within the genomes of P. multocida strains. The annotated phage genomes were analysed since phages are known to carry virulence factors, including genes encoding OMPs and various toxins, and also mediate horizontal DNA transfer. Nucleotide sequence analysis of λ-like phage genomes induced in toxigenic porcine isolates, PM684 and PM848 of capsular types A and D, demonstrated the presence of the toxA gene which encodes the P. multocida toxin (PMT). Moreover, genomic analysis identified additional intact λ-like prophages containing toxA within the bacterial genomes of the porcine toxigenic isolates PM918, PM926, PM40 and PM696 as well as in ovine isolates of capsular type D. No genes encoding OMPs were found to be carried by any of the bacteriophages. Overall, it was concluded that strains of P. multocida recovered from different host species carry a diverse range of bacteriophages. The presence of two bands of different molecular size of phage DNA from isolates PM86, PM172, PM486, PM934 and PM954; together with the identification of small capsids by TEM, suggest that these elements represent PICIs. Interestingly, Southern blot hybridisation of phage DNA in these isolates confirmed induction of both Mu-like phages and PICIs for the first time in P. multocida. This study represents the first comparative genomic analysis of the genes encoding the outer membrane proteome of P. multocida; it also represents the first detailed characterisation of the temperate bacteriophage content of a large number of P. multocida isolates recovered from different host species (cattle, sheep, pigs and poultry) and various disease syndromes. The study has, for the first time, identified PICIs in P. multocida

Molecular characterization of a cytotoxic porin protein from Campylobacter jejuni and its role in campylobacteriosis

Campylobacter jejuni is a major cause of enteritis in developed and developing countries. A cytotoxic complex was isolated from organism-free filtrates of broth grown organisms by screening for activity in HEp-2 cells following high performance liquid chromatography using a combination of size exclusion and DEAE column chromatography. The toxin was found to coincide with a 45 kDa protein possessing an N-terminus sequence indicative of a bacterial outer membrane porin protein together with a high molecular weight carbohydrate which was determined to be lipopolysaccharide (LPS). The carbohydrate portion of the LPS had reactivity for the lectins Galanthus nivalis agglutinin, Maakia amurensis agglutinin and Datura stromonium agglutinin. The cytotoxicity of the porin-LPS complex was heat-labile at 70\sp\circC within 30 min. It was also found to be resistant to trypsin and to degradation by the enzymes neuriminidase and glycosidase F as well as to oxidation with sodium meta-periodate. The complex induced DNA fragmentation, cytoplasmic blebbing and nuclear condensation in HEp-2 cells after 24 h of intoxication possibly indicating that the cells were becoming apoptotic. Oligonucleotides were generated from the N-terminus of the porin protein. Using a combination of vectorette and inverse polymerase chain reaction (PCR) and cloning, a 1.45 kb fragment was sequenced and found to contain a 1275 base pair (bp) open reading frame (ORF). The ORF, designated porA encoded a 424 amino acid protein with a 22 residue leader sequence and had a calculated molecular weight of 45.6 kDa and pI of 4.44. The mature protein was 402 amino acids in length and had a molecular weight of 43.5 kDa and a pI of 4.35. The translated PorA protein had a 50% sequence homology with Haemophilis influenzae major outer membrane protein P2. Thirty-two strains of Campylobacter sp. and closely related organisms were screened for the porA gene and expression of cytotoxin in cell culture. Although all strains examined produced a cytotoxin, 21 of the 32 strains (66%) were genotypically positive for porA and of these 21 of 23 (91%) were C. jejuni strains. It is postulated that this cytotoxic protein is a major virulence factor for C. jejuni and may be responsible for the clinical symptoms usually associated with campylobacteriosis