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Role of Class A Penicillin-Binding Proteins in PBP5-Mediated ß-Lactam Resistance in Enterococcus faecalis. Ana Arbeloa, 2004.Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins [PBP] of classes A and B that associatea conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively . In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of ß-lactam antibiotics,such as ceftriaxone . To identify the glycosyltransferase partner[s]of PBP5, combinations of deletions were introduced in all threeclass A PBP genes of E . faecalis JH2-2 [ponA, pbpF, and pbpZ]. Among mutants with single or double deletions, only JH2-2 deltaponA deltapbpF was susceptible to ceftriaxone . Ceftriaxone resistancewas restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferaseof Staphylococcus aureus . Thus, PBP5 partners essential forpeptidoglycan polymerization in the presence of ß-lactamsformed a subset of the class A PBPs of E . faecalis, and heterospecificcomplementation was observed with an ortholog from E . faecium.Site-directed mutagenesis of pbpF confirmed that the catalyticserine residue of the transpeptidase module was not requiredfor resistance . None of the three class A PBP genes was essentialfor viability, although deletion of the three genes led to anincrease in the generation time and to a decrease in peptidoglycan cross-linking . As the E . faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase . The latter enzyme was not inhibited by moenomycin, since deletion of thethree class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor. The CorA Mg2+ Transporter Is a Homotetramer. Mary Ann Warren, 2004.CorA is a primary Mg2+ transporter for Bacteria and Archaea . The C-terminal domain of  80
amino acids forms three transmembrane (TM) segments, which suggests
that CorA is a homo-oligomer . A Cys residue was added to the
cytoplasmic C terminus (C317) of Salmonella enterica serovar
Typhimurium CorA with or without mutation of the single periplasmic
Cys191 to Ser; each mutant retained function . Oxidation of the
Cys191Ser Cys317 CorA gave a dimer . Oxidation of Cys317 CorA showed a
dimer plus an additional band, apparently cross-linked via both
Cys317 and C191 . To determine oligomer order, intact cells or
purified membranes were treated with formaldehyde or carbon
disulfide . Higher-molecular-mass bands formed, consistent with the
presence of a tetramer . Cross-linking of the Bacillus subtilis
CorA expressed in Salmonella serovar Typhimurium similarly
indicated a tetramer . CorA periplasmic soluble domains from both
Salmonella serovar Typhimurium and the archaeon Methanococcus
jannaschii were purified and shown to retain structure .
Formaldehyde treatment showed formation of a tetramer . Finally,
previous mutagenesis of the CorA membrane domain identified six
intramembrane residues forming an apparent pore that interacts with
Mg2+ during transport . Each was mutated to Cys . In mutants
carrying a single intramembrane Cys residue, spontaneous disulfide
bond formation that was enhanced by oxidation with
Cu(II)-1,10-phenanthroline was observed between monomers, indicating
that these Mg2+-interacting residues within the membrane
are very close to their cognate residue on another monomer . Thus,
CorA appears to be a homotetramer with a TM segment of one monomer
physically close to the same TM segment of another monomer .
Novel Azasterols as Potential Agents for Treatment of Leishmaniasis and Trypanosomiasis. Silvia Orenes Lorente, 2004.This paper describes the design and evaluation of novel azasterols as potential compounds for the treatment of leishmaniasis and other diseases caused by trypanosomatid parasites . Azasterols are a known class of (S)-adenosyl-L-methionine:  24-sterol methyltransferase(24-SMT) inhibitors in fungi, plants, and some parasitic protozoa . The compounds prepared showed activity at micromolar and nanomolar concentrations when tested against Leishmania spp . and Trypanosoma spp . The enzymatic and sterol composition studies indicated that the most active compounds acted by inhibiting 24-SMT . The role of the free hydroxyl group at position 3 of the sterol nucleus was also probed . When an acetate was attached to the 3ß-OH, the compounds did not inhibit the enzyme but had an effect on parasite growth and the levels of sterols in the parasite, suggesting that the acetate group was removed in the organism . Thus, an acetate group on the 3ß-OH may have application as a prodrug . However, there may be an additional mode(s) of action for these acetate derivatives . These compounds were shown to have ultrastructural effects on Leishmania amazonensis promastigote membranes, including the plasma membrane, the mitochondrial membrane, and the endoplasmic reticulum . The compounds were also found to be active against the bloodstream form (trypomastigotes) of Trypanosoma brucei rhodesiense, a causative agent of African trypanosomiasis .
The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route. Sinda Fedhila, 2002.The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts . We identified a second copy of the inhA gene, named inhA2, in B . thuringiensis strain 407 Cry- . The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases . We used a transcriptional inhA2'-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background . The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2 . To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes . Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route . Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel . We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway . The spores of  inhA2 mutant strain were ineffective in providing synergism whereas those of the
inhA mutant strain were not . These results indicate that the B . thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route .
A Gene Encoding a Homologue of Dolichol Phosphate-ß-D-Mannose Synthase Is Required for Infection of Streptomyces coelicolor A3(2) by Phage  C31.Deborah A. Cowlishaw, 2002.We have shown previously that a gene encoding a homologue to the eukaryotic dolichol-phosphate-D-mannose, protein O-D-mannosyltransferase, was required for C31 infection of Streptomyces coelicolor . Here we show that a gene encoding the homologue to dolichol-phosphate-mannose synthase is also essential for phage sensitivity . These data confirm the role of glycosylation in the phage receptor for
C31 in S . coelicolor .
Morphological, Host Range, and Genetic Characterization of Two Coliphages. Lawrence Goodridge, 2003.Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties . Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length . Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length . Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria . The results showed that both phages could infect many serotypes of E . coli . Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages . The genetic material of AR1 and LG1 was characterized . Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1 . Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages . The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking . Southern hybridizations showed that AR1 and T4 are genetically related . The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E . coli in animals and food .
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