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Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential. Hope Richard, 2004.Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2 . One acid resistance system, acid resistance system 2 [AR2], requires extracellular glutamate, while another[AR3] requires extracellular arginine . Little is known abouthow these systems protect cells from acid stress . AR2 and AR3are thought to consume intracellular protons through amino aciddecarboxylation . Antiport mechanisms then exchange decarboxylationproducts for new amino acid substrates . This form of protonconsumption could maintain an internal pH [pHi] conducive tocell survival . The model was tested by estimating the pHi andtransmembrane potential [   ]
of cells acid stressed at pH 2.5.During acid challenge, glutamate-
and arginine-dependent systemselevated pHi from 3.6 to
4.2 and 4.7, respectively . However,when pHi was
manipulated to 4.0 in the presence or absence ofglutamate, only
cultures challenged in the presence of glutamatesurvived, indicating
that a physiological parameter aside frompHi was also
important . Measurements of
indicated that aminoacid-dependent acid resistance systems help
convert membranepotential from an inside negative to inside positive
charge,an established acidophile strategy used to survive extreme
acidicenvironments . Thus, reversing
may be a more important acidresistance strategy than maintaining a
specific pHi value.
Proteomic Analysis of Azole Resistance in Candida albicans Clinical Isolates. Massoumeh Z. Hooshdaran, 2004.Changes in protein expression within a matched set of Candida albicans isolates representing the acquisition of azole resistance were examined by two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting . Proteins differentially expressed in association with azole resistance included Grp2p, Ifd1p, Ifd4p, Ifd5p, and Erg10p, a protein involved in the ergosterol biosynthesis pathway . The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY. Anja Schlegel, 2002.The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins . The system is positively regulated by MalT, its transcriptional activator . MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins . MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity . By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK . This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain . Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors . The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them . Some of them became nearly completely resistant to Aes while still being sensitive to MalY . These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT . Identification of an Akinete Marker Gene in Anabaena variabilis. Ruanbao Zhou, 2002.Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium . Because no akinete marker gene has been identified, akinete formation has been little studied genetically . We report the first identification of an akinete marker gene . Sequence Diversity of Treponema pallidum subsp . pallidum tprK in Human Syphilis Lesions and Rabbit-Propagated Isolates. Rebecca E. LaFond, 2003.The tprK gene of Treponema pallidum subsp . pallidum, the causative agent of venereal syphilis, belongs to a 12-member gene family and encodes a protein with a predicted cleavable signal sequence and predicted transmembrane domains . Except for the Nichols type strain, all rabbit-propagated isolates of T . pallidum examined thus far are comprised of mixed populations of organisms with heterogeneous tprK sequences . We show that tprK sequences in treponemes obtained directly from syphilis patients are also heterogeneous . Clustering analysis demonstrates that primary chancre tprK sequences are more likely to cluster within a sample than among samples and that tighter clustering is seen within chancre samples than within rabbit-propagated isolates . Closer analysis of tprK sequences from a rabbit-propagated isolate reveals that individual variable regions have different levels of diversity, suggesting that variable regions may have different intrinsic rates of sequence change or may be under different levels of selection . Most variable regions show increased sequence diversity upon passage . We speculate that the diversification of tprK during infection allows organisms to evade the host immune response, contributing to reinfection and persistent infection . Atypical 16S rRNA Gene Copies in Ochrobactrum intermedium Strains Reveal a Large Genomic Rearrangement by Recombination between rrn Copies. Corinne Teyssier, 2003.Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria . The 16S rDNA sequences of nine O . intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp . available in data banks . Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA . The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule . Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion . Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone . Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion . In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone . Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies . The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies . Purification and Functional Characterization of a Novel  -L-Arabinofuranosidase from Bifidobacterium longum B667.Abelardo Margolles, 2003.The gene encoding a novel -L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced . The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with
-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases . Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism . A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter . The enzyme was purified by nickel affinity chromatography . The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure . AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70°C), and it had an optimum pH of 6.0 and an optimum temperature of 45°C . The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55°C . Chelating and reducing agents did not have any effect on its activity, but the presence of Cu2+, Hg2+, and Zn2+ markedly reduced enzymatic activity . The protein exhibited a high level of activity with p-nitrophenyl
-L-arabinofuranoside, with apparent Km and Vmax values of 0.295 mM and 417 U/mg, respectively . AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose . No endoarabinanase activity was detected . These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides .
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