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A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products. Kendy K. Y. Wong, 2004.The PrfA protein of Listeria monocytogenes functions as a key regulatory factor for the coordinated expression of many virulence genes during bacterial infection of host cells . PrfA activityis controlled by multiple regulatory mechanisms, including anapparent requirement for either the presence of a cofactor orsome form of posttranslational modification that regulates theactivation of PrfA . In this study, we describe the identificationand characterization of a novel PrfA mutation that results inconstitutive activation of the PrfA protein . The PrfA L140Fmutation was found to confer high-level expression of PrfA-regulatedgenes and to be functionally dominant over the wild-type allele.The presence of the PrfA L140F mutation resulted in the aggregationof L . monocytogenes in broth culture and, unlike previouslydescribed prfA mutations, appeared to be slightly toxic to thebacteria . High-level PrfA-dependent gene expression showed noadditional increase in L . monocytogenes strains containing anadditional copy of prfA L140F despite a >4-fold increasein PrfA protein levels . In contrast, the introduction of multiplecopies of the wild-type prfA allele to L . monocytogenes resultedin a corresponding increase in PrfA-dependent gene expression,although overall expression levels remained far below thoseobserved for PrfA L140F strains . These results suggest a hierarchyof PrfA regulation, such that the relative levels of PrfA proteinpresent within the cell correlate with the levels of PrfA-dependentgene expression when the protein is not in its fully activatedstate; however, saturating levels of the protein are then quicklyreached when PrfA is converted to its active form . Regulationof the PrfA activation status must be an important facet ofL . monocytogenes survival, as mutations that result in constitutivePrfA activation may have deleterious consequences for bacterialphysiology. Microarray-Based Analysis of the Staphylococcus aureus  B
Regulon.Markus Bischoff, 2004.Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by B
activity . While
B
was found to positively control 198 genes by a factor of
 2
in at least two of the three genetic lineages analyzed, 53 ORFs were
repressed in the presence of
B .
Gene products that were found to be influenced by
B
are putatively involved in all manner of cellular processes,
including cell envelope biosynthesis and turnover, intermediary
metabolism, and signaling pathways . Most of the genes and/or operons
identified as upregulated by
B
were preceded by a nucleotide sequence that resembled the
B
consensus promoter sequence of Bacillus subtilis . A
conspicuous number of virulence-associated genes were identified as
regulated by
B
activity, with many adhesins upregulated and prominently represented
in this group, while transcription of various exoproteins and toxins
were repressed . The data presented here suggest that the
B
of S . aureus controls a large regulon and is an important
modulator of virulence gene expression that is likely to act
conversely to RNAIII, the effector molecule of the agr locus .
We propose that this alternative transcription factor may be of
importance for the invading pathogen to fine-tune its virulence
factor production in response to changing host environments .
Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water. Isabelle Villena, 2004.Several recent outbreaks of toxoplasmosis were related to drinking water . We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S . Environmental Protection Agency method with the same sample . Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient . Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts . In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases . The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source . PCR was always more sensitive than mouse inoculation . This detection strategy was then applied to 139 environmental water samples collected over a 20-month period . Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition . Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%) . None of the samples were positive by mouse inoculation . This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method . Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II. Olaf Degen, 2002.HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family . These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI . Two histidine residues in a Ni2+ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity . Replacement of the corresponding His residues in NhlF affected both Co2+ and Ni2+ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations . Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF) . Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important . The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+ uptake significantly . Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+ and Co2+ with low capacity . The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+ uptake capacity and conferred to the variant the ability to transport Co2+ . NhlF activity dropped in response to the converse mutation . Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter . As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity . Identification and Characterization of the Nickel Uptake System for Urease Biogenesis in Streptococcus salivarius 57.I. Yi-Ywan M. Chen, 2003.Ureases are multisubunit enzymes requiring Ni2+ for activity . The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes) . Urease biogenesis also requires a high-affinity Ni2+ uptake system . By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon . To determine whether these genes were involved in urease biogenesis by catalyzing Ni2+ uptake in S . salivarius, regions 3' to ureD were amplified by PCRs from S . salivarius by using primers identical to the S . thermophilus sequences . Sequence analysis of the products revealed three ORFs . Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon . Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate 63Ni2+ during growth . Supplementation of the growth medium with NiCl2 at concentrations as low as 2.5 µM partially restored urease activity in the mutant . Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni2+ was provided at neutral pH . Enhancement of urease activity by adding nickel was regulated at the posttranslational level . Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni2+-specific ATP-binding cassette transporter . Purification and Characterization of an Inverting Stereo- and Enantioselective sec-Alkylsulfatase from the Gram-Positive Bacterium Rhodococcus ruber DSM 44541. Mateja Pogorevc, 2003.Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (±)-2-octyl sulfate in a stereo- and enantiospecific fashion . When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol . From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification . In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa . Maximal enzyme activity was observed at 30°C and at pH 7.5 . Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]) . Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom . Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C7 to C10) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates . Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X) .
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