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Multiple-Locus Variable-Number Tandem Repeat Analysis of Dutch Bordetella pertussis Strains Reveals Rapid Genetic Changes with Clonal Expansion during the Late 1990s. Leo M. Schouls, 2004.Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953 . In the 1990s, several epidemic periods have resulted in many cases of pertussis . We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands . Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology . For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B . pertussis . By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains . The relationships between the various genotypes were visualized by constructing a minimum spanning tree . MLVA of Dutch strains of B . pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s . Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods . Using this genotyping, we have been able to show that B . pertussis is much more dynamic than expected . In Vitro Interactions between Amphotericin B, Itraconazole, and Flucytosine against 21 Clinical Aspergillus Isolates Determined by Two Drug Interaction Models. D. T. A. Te Dorsthorst, 2004.Combination therapy of flucytosine (5FC) with other antifungal agents could be of use for the treatment of invasive aspergillosis . However, interpretation of the results of in vitro interactions is problematic . The fractional inhibitory concentration (FIC) index is the most commonly used method, but it has several major drawbacks in characterizing antifungal drug interaction . Alternatively, a response surface approach using the concentration-effect relationship over the whole concentration range instead of just the MIC can be used . We determined the in vitro interactions between amphotericin B (AMB), itraconazole, and 5FC against 21 Aspergillus isolates with a broth microdilution checkerboard method that employs the dye MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] . FIC indices based on three different MIC endpoints (MIC-0, MIC-1, and MIC-2) and the interaction coefficient alpha were determined, the latter by estimation from the response surface approach described by Greco et al . (W . R . Greco, G . Bravo, and J . C . Parsons, Pharmacol . Rev . 47:331-385, 1995) . The value obtained for the FIC index was found to be dependent on the MIC endpoint used and could be either synergistic, indifferent, or antagonistic . The response surface approach gave more consistent results . Of the three combinations tested, the AMB-5FC combination was the most potent in vitro against Aspergillus spp . We conclude that the use of the response surface approach for the interpretation of in vitro interaction studies of antifungals may be helpful in order to predict the nature and intensity of the drug interaction . However, the correlation of these results with clinical outcome remains difficult and needs to be further investigated . Modification of Spatial Distribution of 2,4-Dichlorophenoxyacetic Acid Degrader Microhabitats during Growth in Soil Columns. C. Pallud, 2004.Bacterial processes in soil, including biodegradation, require contact between bacteria and substrates . Knowledge of the three-dimensional spatial distribution of bacteria at the microscale is necessary to understand and predict such processes . Using a soil microsampling strategy combined with a mathematical spatial analysis, we studied the spatial distribution of 2,4-dichlorophenoxyacetic acid (2,4-D) degrader microhabitats as a function of 2,4-D degrader abundance . Soil columns that allowed natural flow were percolated with 2,4-D to increase the 2,4-D degrader abundance . Hundreds of soil microsamples (minimum diameter, 125 µm) were collected and transferred to culture medium to check for the presence of 2,4-D degraders . Spatial distributions of bacterial microhabitats were characterized by determining the average size of colonized soil patches and the average number of patches per gram of soil . The spatial distribution of 2,4-D degrader microhabitats was not affected by water flow, but there was an overall increase in colonized patch sizes after 2,4-D amendment; colonized microsamples were dispersed in the soil at low 2,4-D degrader densities and clustered in patches that were more than 0.5 mm in diameter at higher densities . During growth, spreading of 2,4-D degraders within the soil and an increase in 2,4-D degradation were observed . We hypothesized that spreading of the bacteria increased the probability of encounters with 2,4-D and resulted in better interception of the degradable substrate . This work showed that characterization of bacterial microscale spatial distribution is relevant to microbial ecology studies . It improved quantitative bacterial microhabitat description and suggested that sporadic movement of cells occurs . Furthermore, it offered perspectives for linking microbial function to the soil physicochemical environment . Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease. Samantha S. Orchard, 2004.The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects . Presently, it is not known what nutrients the bacterium uses to thrive in these host environments . In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X . nematophila in laboratory or host environments . We identified an X . nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF . Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease . We constructed strains with mutations in oppA1, oppA2, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts . We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects . However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions . These data demonstrate that the opp genes allow X . nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X . nematophila in either of its host niches . To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction . FleQ, the Major Flagellar Gene Regulator in Pseudomonas aeruginosa, Binds to Enhancer Sites Located Either Upstream or Atypically Downstream of the RpoN Binding Site. Jeevan Jyot, 2002.In Pseudomonas aeruginosa, flagellar genes are regulated in a cascade headed by FleQ, an NtrC/NifA-type activator . FleQ and RpoN positively regulate expression of flhA, fliE, fliL, and fleSR genes, among others . Direct interaction of FleQ with flhA, fliE, fliL, and fleSR promoters was demonstrated by gel shift assay, along with experiments to conclusively determine the specificity of its binding . DNase I footprinting was performed to determine the FleQ binding sites on flhA, fliE, fliL, and fleSR promoters . No sequence conservation among these binding sites was observed . Primer extension analysis revealed the transcription start sites (TSSs) to be localized above the FleQ binding sites in flhA, fliE, and fliL promoters . Analysis of the above data revealed FleQ binding to be in the leader sequence of these promoters, whereas FleQ binding was 67 bp upstream of the TSS in the fleSR promoter . Mutagenesis of the FleQ binding site in the flhA promoter confirmed its functionality in vivo . Deletion of the flhA promoter upstream of the RNA polymerase binding site did not result in a significant loss of promoter activity . These results point to two modes of regulation by an NtrC-type regulator in the flagellar hierarchy in P . aeruginosa, the first being the typical model of activation from a distance via looping in the fleSR promoter and the second involving flhA, fliE, and fliL promoters, where FleQ binds in the downstream vicinity of the promoter and activates transcription without looping . Rhodospirillum rubrum Possesses a Variant of the bchP Gene, Encoding Geranylgeranyl-Bacteriopheophytin Reductase. Hugh A. Addlesee, 2002.The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe) . In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only . We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function . Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol . We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium . The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2 . In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant . Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites. Dhruva Bhattacharya, 2003.The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined . TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil . The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India . All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing . Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings . Of the isolates, 29 strains of P . citronellolis were selected for evaluating their genetic diversity . This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping . Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies . The 29 strains of P . citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping . The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites . These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P . citronellolis .
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