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CARB-9, a Carbenicillinase Encoded in the VCR Region of Vibrio cholerae Non-O1, Non-O139 Belongs to a Family of Cassette-Encoded ß-Lactamases. Alejandro Petroni, 2004.The gene blaCARB-9 was located in the Vibrio cholerae super-integron, but in a different location relative to blaCARB-7 . CARB-9 (pI 5.2) conferred ß-lactam MICs four to eight times lower than those conferred by CARB-7, differing at Ambler's positions V97I, L124F, and T228K . Comparison of the genetic environments of all reported blaCARB genes indicated that the CARB enzymes constitute a family of cassette-encoded ß-lactamases . Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in "Streptomyces carzinostaticus". Miyuki Otsuka, 2004.Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by "Streptomyces carzinostaticus" var . F-41 . The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized . DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules . Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module . Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B . Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis . Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7 . This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain . Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites . The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite . Combining Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization and Microautoradiography To Detect Substrate Utilization by Bacteria and Archaea in the Deep Ocean. Eva Teira, 2004. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein. Francisca Reyes-Ramirez, 2002.The Azotobacter vinelandii  54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status . Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex . NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by
54-RNA polymerase and may also inhibit the activator-polymerase interaction . To analyze the mechanism of inhibition in greater detail, we isolated NifA mutants which are resistant to the inhibitory action of NifL . Mutations in both the amino-terminal GAF domain and the catalytic AAA+ domain of NifA were isolated . Several mutants blocked inhibition by NifL in response to both nitrogen and redox status, whereas some of the mutant NifA proteins were apparently able to discriminate between the forms of NifL present under different environmental conditions . One mutant protein, NifA-Y254N, was resistant to NifL under conditions of anaerobic nitrogen excess but was relatively sensitive to NifL under aerobic growth conditions . The properties of the purified mutant protein in vitro were consistent with the in vivo phenotype and indicate that NifA-Y254N is not responsive to the nitrogen signal conveyed by the interaction of NifL with A . vinelandii GlnK but is responsive to the oxidized form of NifL when ADP is present . Our observations suggest that different conformers of NifL may be generated in response to discrete signal transduction events and that both the GAF and AAA+ domains of NifA are involved in the response to NifL .
Novel Type of Specialized Transduction for CTX  or Its Satellite Phage RS1 Mediated by Filamentous Phage VGJ in Vibrio cholerae.Javier Campos, 2003.The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX . This phage
transmits ctxAB genes between V . cholerae bacterial
populations that express toxin-coregulated pilus (TCP), the
CTX receptor . In investigating new forms of ctxAB
transmission, we found that V . cholerae filamentous phage
VGJ, which uses the mannose-sensitive hemagglutinin (MSHA)
pilus as a receptor, transmits CTX or its satellite phage RS1
by an efficient and highly specific TCP-independent mechanism . This is
a novel type of specialized transduction consisting in the
site-specific cointegration of VGJ and CTX (or RS1)
replicative forms to produce a single hybrid molecule, which generates
a single-stranded DNA hybrid genome that is packaged into hybrid viral
particles designated HybP (for the VGJ/CTX
hybrid) and HybRS (for the VGJ/RS1 hybrid) . The
hybrid phages replicate by using the VGJ replicating functions
and use the VGJ capsid, retaining the ability to infect via
MSHA . The hybrid phages infect most tested strains more efficiently
than CTX, even under in vitro optimal conditions for TCP
expression . Infection and lysogenization with HybP revert the
V . cholerae live attenuated vaccine strain 1333 to virulence.
Our results reinforce that TCP is not indispensable for the acquisition
of CTX . Thus, we discuss an alternative to the current
accepted evolutionary model for the emergence of new toxigenic strains
of V . cholerae and the importance of our findings for the
development of an environmentally safer live attenuated cholera
vaccine .
Purification and Genetic Characterization of Plantaricin NC8, a Novel Coculture-Inducible Two-Peptide Bacteriocin from Lactobacillus plantarum NC8. Antonio Maldonado, 2003.A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63 . This bacteriocin consists of two distinct peptides, named  and ß, which were separated by C2-C18 reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity . N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 and PLNC8ß, respectively, which showed no sequence similarity to other known bacteriocins . Mass spectrometry analysis showed molecular masses of 3,587 Da () and 4,000 Da (ß) . The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type . The mature
and ß peptides contain 29 and 34 amino acids, respectively . An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A . Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism .
Identification of Intermediate and Branch Metabolites Resulting from Biotransformation of 2-Benzoxazolinone by Fusarium verticillioides. A. E. Glenn, 2003.Detoxification of the maize (Zea mays) antimicrobial compound 2-benzoxazolinone by the fungal endophyte Fusarium verticillioides involves two genetic loci, FDB1 and FDB2, and results in the formation of N-(2-hydroxyphenyl)malonamic acid . Intermediate and branch metabolites were previously suggested to be part of the biotransformation pathway . Evidence is presented here in support of 2-aminophenol as the intermediate metabolite and 2-acetamidophenol as the branch metabolite, which was previously designated as BOA-X . Overall, 2-benzoxazolinone metabolism involves hydrolysis (FDB1) to produce 2-aminophenol, which is then modified (FDB2) by addition of a malonyl group to produce N-(2-hydroxyphenyl)malonamic acid . If the modification is prevented due to genetic mutation (fbd2), then 2-acetamidophenol may accumulate as a result of addition of an acetyl group to 2-aminophenol . This study resolves the overall chemistry of the 2-benzoxazolinone detoxification pathway, and we hypothesize that biotransformation of the related antimicrobial 6-methoxy-2-benzoxazolinone to produce N-(2-hydroxy-4-methoxyphenyl)malonamic acid also occurs via the same enzymatic modifications . Detoxification of these antimicrobials by F . verticillioides apparently is not a major virulence factor but may enhance the ecological fitness of the fungus during colonization of maize stubble and field debris .
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