|
|
|
|
|
|
The ttsA Gene Is Required for Low-Calcium-Induced Type III Secretion of Yop Proteins and Virulence of Yersinia enterocolitica W22703. Kristin L. DeBord, 2003.Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues . At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals . Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins . To identify such genes, we screened transposon insertion mutants of Y . enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein . ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins . ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes . All of these defects were restored by complementation with plasmid-encoded wild-type ttsA . LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal . Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone . To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice . The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain . Nucleotide and Amino Acid Polymorphisms at Drug Resistance Sites in Non-B-Subtype Variants of Human Immunodeficiency Virus Type 1. Dan Turner, 2004.We have compared nucleotide substitutions and polymorphisms at codons known to confer drug resistance in subtype B strains of human immunodeficiency virus type 1 (HIV-1) with similar substitutions in viruses of other subtypes . Genotypic analysis was performed on viruses from untreated individuals . Nucleotide and amino acid diversity at resistance sites was compared with a consensus subtype B reference virus . Among patients with non-subtype B infections, polymorphisms relative to subtype B were observed at codon 10 in protease (PR) . These included silent substitutions (CTC  CTT, CTA, TTA) and an amino acid mutation, L10I . Subtype A viruses possessed a V179I substitution in reverse transcriptase (RT) . Subtype G viruses were identified by silent substitutions at codon 181 in RT (TATTAC) . Similarly, subtype A/G viruses were identified by a substitution at position 67 in RT (GACGAT) . Subtype C was distinguished by silent substitutions at codons 106 (GTAGTG) and 219 (AAAAAG) in RT and codon 48 (GGGGGA) in PR . Variations relative to subtype B were seen at RT position 215 (ACCACT) for subtypes A and A/E . These substitutions and polymorphisms reflect different patterns of codon usage among viruses of different subtypes . However, the existence of different subtypes may only rarely affect patterns of drug resistance-associated mutations .
Accurate Estimation of Viral Abundance by Epifluorescence Microscopy. Kevin Wen, 2004.Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples . Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM . Our results indicate that aldehyde fixation results in rapid decreases in viral abundance . By 1 h postfixation, the abundance dropped by 16.4% ± 5.2% (n = 6), and by 4 h, the abundance was 20 to 35% lower . The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h–1, respectively . By 16 days, viral abundance had decreased by 72% (standard deviation, 6%; n = 6) . Aldehyde fixation of samples followed by storage at 4°C, for even a few hours, resulted in large underestimates of viral abundance . The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h–1 during the first hour to 0.01 h–1 between 24 and 48 h . Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h . Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at –20°C for at least 2 weeks or, for Yo-Pro-1, at least 1 year . If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at –86°C . Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen . If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance . Identification by Flagellum Display of an Epithelial Cell- and Fibronectin-Binding Function in the SlpA Surface Protein of Lactobacillus brevis. Ulla Hynönen, 2002.Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin . For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliCH7 gene of Escherichia coli . The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size . The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence . Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella . The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein . The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin . The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells . In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells . The results identify the S-layer of L . brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies . Expression, Purification, and Characterization of AknX Anthrone Oxygenase, Which Is Involved in Aklavinone Biosynthesis in Streptomyces galilaeus. Jin-young Chung, 2002.In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes . aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus . In order to identify its function, the aknX gene was expressed in Escherichia coli . The cell extract prepared from E . coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin . This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6 . The AknX protein fused with a His6 tag was efficiently purified to homogeneity by Ni2+ affinity and anion-exchange column chromatography . The native molecular mass of AknX was estimated to be 42 kDa by gel filtration . Thus, native AknX is considered to have a homotrimeric subunit structure . AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation . Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction . Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction . A possible mechanism for the reaction via peroxy anion intermediate is proposed .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
