Identification of Novel Pathogenicity Loci in Clostridium perfringens Strains That Cause Avian Necrotic Enteritis

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne

A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

<p>Abstract</p> <p>Background</p> <p>tRNase Z removes the 3'-trailer sequences from precursor tRNAs, which is an essential step preceding the addition of the CCA sequence. tRNase Z exists in the short (tRNase Z^S) and long (tRNase Z^L) forms. Based on the sequence characteristics, they can be divided into two major types: bacterial-type tRNase Z^Sand eukaryotic-type tRNase Z^L, and one minor type, <it>Thermotoga maritima </it>(TM)-type tRNase Z^S. The number of tRNase Zs is highly variable, with the largest number being identified experimentally in the flowering plant <it>Arabidopsis thaliana</it>. It is unknown whether multiple tRNase Zs found in <it>A. thaliana </it>is common to the plant kingdom. Also unknown is the extent of sequence and structural conservation among tRNase Zs from the plant kingdom.</p> <p>Results</p> <p>We report the identification and analysis of candidate tRNase Zs in 27 fully sequenced genomes of green plants, the great majority of which are flowering plants. It appears that green plants contain multiple distinct tRNase Zs predicted to reside in different subcellular compartments. Furthermore, while the bacterial-type tRNase Z^Ss are present only in basal land plants and green algae, the TM-type tRNase Z^Ss are widespread in green plants. The protein sequences of the TM-type tRNase Z^Ss identified in green plants are similar to those of the bacterial-type tRNase Z^Ss but have distinct features, including the TM-type flexible arm, the variant catalytic HEAT and HST motifs, and a lack of the PxKxRN motif involved in CCA anti-determination (inhibition of tRNase Z activity by CCA), which prevents tRNase Z cleavage of mature tRNAs. Examination of flowering plant chloroplast tRNA genes reveals that many of these genes encode partial CCA sequences. Based on our results and previous studies, we predict that the plant TM-type tRNase Z^Ss may not recognize the CCA sequence as an anti-determinant.</p> <p>Conclusions</p> <p>Our findings substantially expand the current repertoire of the TM-type tRNase Z^Ss and hint at the possibility that these proteins may have been selected for their ability to process chloroplast pre-tRNAs with whole or partial CCA sequences. Our results also support the coevolution of tRNase Zs and tRNA 3'-trailer sequences in plants.</p

Using Light to Improve Commercial Value

The plasticity of plant morphology has evolved to maximize reproductive fitness in response to prevailing environmental conditions. Leaf architecture elaborates to maximize light harvesting, while the transition to flowering can either be accelerated or delayed to improve an individual's fitness. One of the most important environmental signals is light, with plants using light for both photosynthesis and as an environmental signal. Plants perceive different wavelengths of light using distinct photoreceptors. Recent advances in LED technology now enable light quality to be manipulated at a commercial scale, and as such opportunities now exist to take advantage of plants' developmental plasticity to enhance crop yield and quality through precise manipulation of a crops' lighting regime. This review will discuss how plants perceive and respond to light, and consider how these specific signaling pathways can be manipulated to improve crop yield and quality

Solution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid

Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present study we describe the high-resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase BcII and of its complex with R-thiomandelic acid, a broad-spectrum inhibitor of metallo-β-lactamases. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active-site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3–β4 loop. Comparisons with other metallo-β-lactamases highlight the roles of individual amino-acid residues in the active site and the β3–β4 loop in inhibitor binding and provide information on the basis of structure–activity relationships among metallo-β-lactamase inhibitors

VIM-2 beta-lactamase in Pseudomonas aeruginosa isolates from Zagreb, Croatia

The aim of this investigation was to characterize metallo-beta-lactamases (MBLs) in Pseudomonas aeruginosa isolates from Zagreb, Croatia. One hundred P. aeruginosa isolates with reduced susceptibility to either imipenem or meropenem were tested for the production of MBLs by MBL-Etest. The susceptibility to a wide range of antibiotics was determined by broth microdilution method. The presence of bla(MBL) genes was detected by polymerase chain reaction (PCR). Hydrolysis of 0.1 mM imipenem by crude enzyme preparations of beta-lactamases was monitored by UV spectrophotometer. Outer membrane proteins were prepared and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Six out of 100 isolates were positive for MBLs by Etest. All strains were resistant to gentamicin, ceftazidime and cefotaxime, and all except 1 were resistant to imipenem. Six strains positive for MBLs by Etest were identified as VIM MBL-producers by PCR. Sequencing of bla(VIM) genes revealed the production of VIM-2 beta-lactamase in all 6 strains. This investigation proved the occurrence of VIM-2 beta-lactamase among P. aeruginosa strains from Zagreb, Croatia. VIM-2 beta-lactamase with similar properties has previously been described in another region of Croatia and in Italy, France, Spain, Greece, Taiwan and South Korea, suggesting that this type of enzyme is widespread in the Mediterranean region of Europe and in the Far East