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The hFbpABC Transporter from Haemophilus influenzae Functions as a Binding-Protein-Dependent ABC Transporter with High Specificity and Affinity for Ferric Iron. Damon S. Anderson, 2004.Pathogenic Haemophilus influenzae, Neisseria spp . [Neisseria gonorrhoeae and N . meningitidis], Serratia marcescens, and othergram-negative bacteria utilize a periplasm-to-cytosol FbpABCiron transporter . In this study, we investigated the H . influenzaeFbpABC transporter in a siderophore-deficient Escherichia colibackground to assess biochemical aspects of FbpABC transporterfunction . Using a radiolabeled Fe3+ transport assay, we establishedan apparent Km = 0.9 µM and Vmax = 1.8 pmol/107cells/min for FbpABC-mediated transport . Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect . Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals . Metal sensitivityexperiments showed that several divalent metals, including copper,nickel, and zinc, exhibited general toxicity towards E . coli.Significantly, gallium-induced toxicity was specific only toE . coli expressing FbpABC . A single-amino-acid mutation in thegene encoding the periplasmic binding protein, FbpA[Y196I],resulted in a greatly diminished iron binding affinity Kd =5.2 x 10–4 M–1, ~14 orders of magnitude weaker thanthat of the wild-type protein . Surprisingly, the mutant transporter[FbpA[Y196I]BC] exhibited substantial transport activity, ~35% of wild-type transport, with Km = 1.2 µM and Vmax = 0.5pmol/107cells/min . We conclude that the FbpABC complexes possessbasic characteristics representative of the family of bacterialbinding protein-dependent ABC transporters . However, the specificityand high-affinity binding characteristics suggest that the FbpABCtransporters function as specialized transporters satisfyingthe strict chemical requirements of ferric iron [Fe3+] bindingand membrane transport. Biotransformation in Double-Phase Systems: Physiological Responses of Pseudomonas putida DOT-T1E to a Double Phase Made of Aliphatic Alcohols and Biosynthesis of Substituted Catechols. Antonia Rojas, 2004.Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logPow (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of  2.5 . Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase . We tested P . putida DOT-T1E tolerance to different aliphatic alcohols with a logPow value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logPow around 1.5 are produced . P . putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons . These defense mechanisms allow P . putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised . Our results support that the logPow of aliphatic alcohols correlates with their toxic effects, as octanol (logPow = 2.9) has more negative effects in P . putida cells than 1-nonanol (logPow = 3.4) or 1-decanol (logPow = 4) . A P . putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logPow = 3.2) into 3-methylcatechol (logPow = 1.8) . The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation .
The ner Gene of Photorhabdus: Effects on Primary-Form-Specific Phenotypes and Outer Membrane Protein Composition. Keith H. O'Neill, 2002.The nematode-bacterium complex of Heterorhabditis-Photorhabdus is pathogenic to insect larvae . The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes . The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries . Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as ner, a regulatory gene in gram-negative bacteria and their phages . Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form . These and other pieces of evidence indicate that the endogenous ner gene is not responsible for the secondary phenotype, but that ner, when overexpressed, can repress expression of primary phenotypes at stationary phase . Inactivation of the endogenous ner gene in the primary form affected the outer membrane protein profile . A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the ner primary strain . Converting the NiFeS Carbon Monoxide Dehydrogenase to a Hydrogenase and a Hydroxylamine Reductase. Jongyun Heo, 2002.Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme . In some cases, however, the specificity of the enzyme is changed . In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase . CODH with alanine substituted for Cys531 exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO . CODH with valine substituted for His265 exhibits hydroxylamine reductase activity . Both Cys531 and His265 are ligands to the active-site cluster of CODH . Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity . Increasing the Ratio of Soj to Spo0J Promotes Replication Initiation in Bacillus subtilis. Yoshitoshi Ogura, 2003.The ParA and ParB protein families are well conserved in bacteria . However, their functions are still unclear . In Bacillus subtilis, Soj and Spo0J are members of these two protein families, respectively . A previous report revealed that replication initiated early and asynchronously in spo0J null mutant cells, as determined by flow cytometry . In this study, we examined the cause of this promotion of replication initiation . Deletion of both the soj and spo0J genes restored the frequency of replication initiation to almost the wild-type level, suggesting that production of Soj in the absence of Spo0J leads to early and asynchronous initiation of replication . Consistent with this suggestion, overproduction of Soj in wild-type cells had the same effect on replication initiation as in the spo0J null mutant, and overproduction of both Soj and Spo0J did not . These results indicate that when the ratio of Soj to Spo0J increases, Soj interferes with tight control of replication initiation and causes early and asynchronous initiation . Whereas replication initiation also occurred significantly earlier in the two spo0J mutants, spo0J14 and spo0J17, it occurred only slightly early in the sojK16Q mutant and was delayed in the sojG12V mutant . Although Soj localized to nucleoids in the spo0J mutants, the two Soj mutant proteins were distributed throughout the cell or localized to cell poles . Thus, interestingly, the promotion of replication initiation seems to correlate with localization of Soj to nucleoids . This may suggest that Soj inhibits transcription of some cell cycle genes and leads to early and asynchronous initiation of replication . In wild-type cells Spo0J counteracts this Soj function . The Cell Wall of the Pathogenic Bacterium Rhodococcus equi Contains Two Channel-Forming Proteins with Different Properties. Franziska G. Rieß, 2003.We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties . The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions . The channel-forming protein PorAReq (R . equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated . PorAReq has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges . According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa . The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm . The second channel (formed by PorBReq [R . equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated . This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth . Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa . Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes . We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales . Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of Bacillus and Related Genera. Daniele Daffonchio, 2003.The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels . We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus . We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices . The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS . We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles . Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing .
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