scholarly journals Telithromycin Resistance in Streptococcus pneumoniae Is Conferred by a Deletion in the Leader Sequence of erm(B) That Increases rRNA Methylation

2007 ◽  
Vol 52 (2) ◽  
pp. 435-440 ◽  
Author(s):  
Nicole Wolter ◽  
Anthony M. Smith ◽  
David J. Farrell ◽  
John Blackman Northwood ◽  
Stephen Douthwaite ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Leader Sequence ◽  
Lag Phase ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Resistant Phenotype ◽  
Type Gene ◽  
Rrna Methylation

ABSTRACT A telithromycin-resistant clinical isolate of Streptococcus pneumoniae (strain P1501016) has been found to contain a version of erm(B) that is altered by a 136-bp deletion in the leader sequence. By allele replacement mutagenesis, a second strain of S. pneumoniae (PC13) with a wild-type erm(B) gene was transformed to the telithromycin-resistant phenotype by introduction of the mutant erm(B) gene. Whereas the wild-type PC13 strain showed slight telithromycin resistance only after induction by erythromycin (telithromycin MIC increased from 0.06 to 0.5 μg/ml), the transformed PC13 strain is constitutively resistant (MIC of 16 μg/ml). Expression of erm(B) was quantified by real-time reverse transcription-PCR in the presence of erythromycin or telithromycin; erm(B) expression was significantly higher in the transformed PC13 strain than the wild-type strain. Furthermore, the transformed strain had significantly higher levels of ribosomal methylation in the absence as well as in the presence of the antibiotics. Growth studies showed that the transformed PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance is conclusively shown to be conferred by the mutant erm(B) gene that is expressed at a constitutively higher level than the inducible wild-type gene. Elevated erm(B) expression results in a higher level of rRNA methylation that presumably hinders telithromycin binding to the ribosome.

scholarly journals C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases

2009 ◽  
Vol 8 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Alejandro D. Nusblat ◽  
Sebastián R. Najle ◽  
Mariela L. Tomazic ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
Gene Identification ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Gene Coding ◽  
Transmembrane Regions

ABSTRACT The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

scholarly journals Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

2001 ◽  
Vol 183 (2) ◽  
pp. 528-535 ◽  
Author(s):  
Hsien-Ming Lee ◽  
Shiaw-Wei Tyan ◽  
Wei-Ming Leu ◽  
Ling-Yun Chen ◽  
David Chanhen Chen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mutant Strain ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Wild Type Strain ◽  
Type Ii ◽  
Wild Type ◽  
In The Wild ◽  
Steady State Levels ◽  
Type Gene

ABSTRACT The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestrispv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from thexpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into thexpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsNgene or by introducing extra copies of wild-type xpsL orxpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL orxpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with thexpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of thexpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

scholarly journals Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against Streptococcus pneumoniae

2001 ◽  
Vol 45 (6) ◽  
pp. 1649-1653 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Ryuta Kishii ◽  
Masaya Takei ◽  
Masaki Hosaka
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Methoxy Group ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Mutant Strains

ABSTRACT Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 × 10−9) from wild-type strainStreptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3′-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 × 10−6). The MIC of AM-1147 increased for the gyrA mutant strains but not for theparC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-stepparC and gyrA mutant strains, respectively, and two- to eightfold higher against the second-step gyrA andparC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

scholarly journals Characterization of emeA, anorA Homolog and Multidrug Resistance Efflux Pump, inEnterococcus faecalis

2001 ◽  
Vol 45 (12) ◽  
pp. 3574-3579 ◽  
Author(s):  
Brandie M. Jonas ◽  
Barbara E. Murray ◽  
George M. Weinstock
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Type Gene ◽  
Encoding Genes

ABSTRACT We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

scholarly journals l-Sorbose Reductase and Its Transcriptional Regulator Involved in l-Sorbose Utilization of Gluconobacter frateurii

2007 ◽  
Vol 189 (13) ◽  
pp. 4800-4808 ◽  
Author(s):  
Wichai Soemphol ◽  
Hirohide Toyama ◽  
Duangtip Moonmangmee ◽  
Osao Adachi ◽  
Kazunobu Matsushita
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Flavin Adenine Dinucleotide ◽  
Transcriptional Regulator ◽  
Molecular Size ◽  
Adjacent Gene ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Better Than ◽  
Sorbose Reductase

ABSTRACT Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent d-sorbitol dehydrogenase (SLDH), sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent l-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on l-sorbose, indicating that the SboA enzyme is required for efficient growth on l-sorbose. The sboR mutant grew on l-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on d-sorbitol and that another activator is required for the induction of these genes by d-sorbitol or l-sorbose.

scholarly journals Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B Producer Streptomyces acidiscabies

2009 ◽  
Vol 191 (15) ◽  
pp. 4786-4797 ◽  
Author(s):  
Frank G. Healy ◽  
Kevin P. Eaton ◽  
Prajit Limsirichai ◽  
Joel F. Aldrich ◽  
Alaina K. Plowman ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Type Strain ◽  
Wild Type Strain ◽  
Morphological Differentiation ◽  
Pcr Analysis ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Morphological Defects ◽  
In The Wild ◽  
Streptomyces Acidiscabies

ABSTRACT Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNAleu. sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.

scholarly journals Role of Stress Response Sigma Factor SigG in Mycobacterium tuberculosis

2007 ◽  
Vol 190 (3) ◽  
pp. 1128-1133 ◽  
Author(s):  
Jong-Hee Lee ◽  
Deborah E. Geiman ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sos Response ◽  
Wild Type ◽  
Recognition Sequence ◽  
Reverse Transcription Pcr ◽  
Promoter Recognition ◽  
Microarray Studies

ABSTRACT The sigG gene of Mycobacterium tuberculosis was disrupted by homologous recombination, and the genes regulated by SigG were examined by real-time reverse-transcription PCR and microarray studies. The SigG consensus promoter recognition sequence was identified as GCGNGT-N15-18-CGANCA. A ΔsigG mutant was found to be more resistant to mitomycin C treatment than the wild-type strain, indicating that it may be involved in the SOS response in M. tuberculosis.

scholarly journals The Autolytic Enzyme LytA of Streptococcus pneumoniae Is Not Responsible for Releasing Pneumolysin

2001 ◽  
Vol 183 (10) ◽  
pp. 3108-3116 ◽  
Author(s):  
Priya Balachandran ◽  
Susan K. Hollingshead ◽  
James C. Paton ◽  
David E. Briles
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Primary Role ◽  
Wild Type ◽  
Phase Growth ◽  
Total Proteins ◽  
Extracellular Release ◽  
Extracellular Location ◽  
Phage Lysins

ABSTRACT It was previously proposed that autolysin's primary role in the virulence of pneumococci was to release pneumolysin to an extracellular location. This interpretation came into question when pneumolysin was observed to be released in significant amounts from some pneumococci during log-phase growth, because autolysis was not believed to occur at this time. We have reexamined this phenomenon in detail for one such strain, WU2. This study found that the extracellular release of pneumolysin from WU2 was not dependent on autolysin action. A mutant lacking autolysin showed the same pattern of pneumolysin release as the wild-type strain. Addition of mitomycin C to a growing WU2 culture did not induce lysis, indicating the absence of resident bacteriophages that could potentially harbor lytA-like genes. Furthermore, release of pneumolysin was unaltered by growth in 2% choline, a condition which is reported to inactivate autolysin, as well as most known pneumococcal phage lysins. Profiles of total proteins in the cytoplasm and in the supernatant media supported the hypothesis that release of pneumolysin is independent of pneumococcal lysis. Finally, under some infection conditions, mutations in pneumolysin and autolysin had different effects on virulence.

scholarly journals Role of RegM, a Homologue of the Catabolite Repressor Protein CcpA, in the Virulence of Streptococcus pneumoniae

2002 ◽  
Vol 70 (10) ◽  
pp. 5454-5461 ◽  
Author(s):  
Philippe Giammarinaro ◽  
James C. Paton
Keyword(s):  
Streptococcus Pneumoniae ◽  
Growth Rates ◽  
Type Strain ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Ex Vivo ◽  
Wild Type ◽  
Significant Similarity

ABSTRACT As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both α-glucosidase and β-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.

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