@article {3141, title = {When is a microbial culture "pure"? Persistent cryptic contaminant escapes detection even with deep genome sequencing.}, journal = {mBio}, volume = {4}, year = {2013}, month = {2013 Mar 12}, pages = {e00591-12}, abstract = {

UNLABELLED: Geobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (-400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, a c-type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 10(5) copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at -400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode.

IMPORTANCE: Repeated streaking of isolated colonies on solidified medium remains a common strategy for obtaining pure cultures, especially of difficult-to-cultivate microorganisms such as strict anaerobes. The results presented here demonstrate that verifying the purity of cultures obtained in this manner may be difficult because extremely rare variants can persist, undetectable with even deep genomic DNA sequencing. The only way to ensure that a culture is pure is to cultivate it from an initial single cell, which may be technically difficult for many environmentally significant microbes.

}, keywords = {Coinfection, Electrodes, Genes, Bacterial, Genotype, Geobacter, High-Throughput Nucleotide Sequencing, Microbial Interactions, Polymerase Chain Reaction}, issn = {2150-7511}, doi = {10.1128/mBio.00591-12}, author = {Shrestha, Pravin Malla and Nevin, Kelly P and Shrestha, Minita and Lovley, Derek R} } @article {484, title = {Geobacter uraniireducens sp. nov., isolated from subsurface sediment undergoing uranium bioremediation.}, journal = {Int J Syst Evol Microbiol}, volume = {58}, year = {2008}, month = {2008 May}, pages = {1075-8}, abstract = {A Gram-negative, rod-shaped, motile bacterium, strain Rf4T, which conserves energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation, was isolated from subsurface sediment undergoing uranium bioremediation. The 16S rRNA gene sequence of strain Rf4T matched sequences recovered in 16S rRNA gene clone libraries constructed from DNA extracted from groundwater sampled at the same time as the source sediment. Cells of strain Rf4T were regular, motile rods, 1.2-2.0 microm long and 0.5-0.6 microm in diameter, with rounded ends. Cells had one lateral flagellum. Growth was optimal at pH 6.5-7.0 and 32 degrees C. With acetate as the electron donor, strain Rf4T used Fe(III), Mn(IV), anthraquinone-2,6-disulfonate, malate and fumarate as electron acceptors and reduced U(VI) in cell suspensions. With poorly crystalline Fe(III) oxide as the electron acceptor, strain Rf4T oxidized the following electron donors: acetate, lactate, pyruvate and ethanol. Phylogenetic analysis of the 16S rRNA gene sequence of strain Rf4T placed it in the genus Geobacter. Strain Rf4T was most closely related to {\textquoteright}Geobacter humireducens{\textquoteright} JW3 (95.9 \% sequence similarity), Geobacter bremensis Dfr1T (95.4 \%) and Geobacter bemidjiensis BemT (95.1 \%). Based on phylogenetic analysis and phenotypic differences between strain Rf4T and closely related Geobacter species, this strain is described as a representative of a novel species, Geobacter uraniireducens sp. nov. The type strain is Rf4T (=ATCC BAA-1134T =JCM 13001T).}, keywords = {Bacterial Typing Techniques, DNA, Bacterial, Genes, rRNA, Genotype, Geobacter, Geologic Sediments, Molecular Sequence Data, Oxidation-Reduction, Phenotype, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Species Specificity, Uranium}, issn = {1466-5026}, doi = {10.1099/ijs.0.65377-0}, author = {Shelobolina, Evgenya S and Vrionis, Helen A and Findlay, Robert H and Lovley, Derek R} } @article {771, title = {An expectation maximization approach to estimate malaria haplotype frequencies in multiply infected children.}, journal = {Stat Appl Genet Mol Biol}, volume = {6}, year = {2007}, month = {2007}, pages = {Article33}, abstract = {Characterizing genetic variability in the human pathogenic Plasmodium species, the group of parasites that cause Malaria, may have broad global health implications. Specifically, discerning the combinations of mutations that lead to viable parasites and the population level frequencies of these clonal sequences will allow for targeted vaccine development and individualized treatment choices. This presents an analytical challenge, however, since haplotypic phase (i.e. the alignment of bases on a single DNA strand) is generally unobservable in multiply infected individuals. This manuscript describes an expectation maximization (EM) approach to maximum likelihood estimation of haplotype frequencies in this missing data setting. The approach is applied to a cohort of N=341 malaria infected children in Uganda, Cameroon and Sudan to characterize regional differences. A simulation study is also presented to characterize method performance and assess sensitivity to distributional assumptions.}, keywords = {Animals, Child, Gene Frequency, Genotype, Humans, Likelihood Functions, Malaria, Malaria Vaccines, Mutation, Plasmodium}, issn = {1544-6115}, doi = {10.2202/1544-6115.1321}, author = {Li, Xiaohong and Foulkes, Andrea S and Yucel, Recai M and Rich, Stephen M} } @article {782, title = {Experimental evidence for genetic recombination in the opportunistic pathogen Cryptosporidium parvum.}, journal = {Mol Biochem Parasitol}, volume = {119}, year = {2002}, month = {2002 Jan}, pages = {55-62}, abstract = {Cryptosporidium parvum is an intracellular protozoan parasite causing intestinal malabsorption and diarrhea in humans. The infection is usually self-limiting, although persistent cryptosporidosis is observed in immunocompromised and malnourished individuals. As with other Apicomplexa, the life cycle of Cryptosporidium is thought to comprise a sexual phase, during which a motile microgamont fuses with a sessile macrogamont. The four sporozoites found within each oocyst (the infectious form excreted in the feces) are thought to be the product of a meiotic division taking place immediately following fertilization, but the existence of a meiotic cycle in this genus has not been tested experimentally. To substantiate the occurrence of meiotic recombination in this species, we performed a genetic cross between two distinct isolates of C. parvum co-infected in INF-gamma knockout mice. We found that mixed infections produced recombinant progeny characterized by multilocus genotypes comprising alleles inherited from each parental line. This observation represents the first demonstration of sexual recombination in this pathogen. Together with the occurrence of genetically heterogeneous infections, this finding suggests that outcrossing between genotypes may occur in nature. Experimental crosses among Cryptosporidium populations will facilitate mapping of clinically relevant genes, the delineation of Cryptosporidium species, and defining the taxonomical status of C. parvum subtypes and host-specific genotypes.}, keywords = {Animals, Conserved Sequence, Crosses, Genetic, Cryptosporidiosis, Cryptosporidium parvum, Deer, Female, Genotype, Humans, Male, Mice, Mice, Knockout, Microsatellite Repeats, Opportunistic Infections, Polymerase Chain Reaction, Recombination, Genetic, Survival Rate}, issn = {0166-6851}, author = {Feng, Xiaochuan and Rich, Stephen M and Tzipori, Saul and Widmer, Giovanni} } @article {825, title = {PriA mutations that affect PriA-PriC function during replication restart.}, journal = {Mol Microbiol}, volume = {41}, year = {2001}, month = {2001 Aug}, pages = {697-704}, abstract = {In Escherichia coli, repair and restart of collapsed replication forks is thought to be essential for cell growth. The replication restart proteins, PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT and Rep, form redundant pathways that recognize repaired replication forks and restart them. Recognition, modulation of specific DNA structures and loading of the replicative helicase by the replication restart proteins, is likely to be important for replication restart. It has been hypothesized that PriB and PriC function with PriA in genetically separate and redundant PriA-PriB and PriA-PriC pathways. In this study, the del(priB)302 or priC303:kan mutations were used to isolate the PriA-PriB and PriA-PriC pathways genetically so that the effects of three priA missense mutations, priA300 (K230R), priA301 (C479Y) and priA306 (L557P), on these pathways could be assessed. In a wild-type background, the three priA mutations had little, if any, effect on the phenotypes of UV resistance, basal levels of SOS expression and cell viability. In the priB mutant, priA300 and priA301 caused dramatic negative changes in the three phenotypes listed above (and others), whereas the third priA mutant allele, priA306, showed very little negative effect. In the priC mutant, all three priA mutations behaved similarly, producing little, if any, changes in phenotypes. We conclude that priA300 and priA301 mostly affect the PriA-PriC pathway and do so more than priA306. We suggest that PriA{\textquoteright}s helicase activity is important for the PriA-PriC pathway of replication restart.}, keywords = {Bacterial Proteins, Bacteriophage mu, Cell Division, DNA Replication, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Lethal, Genotype, Models, Biological, Mutation, Missense, Phenotype, Replication Protein A, SOS Response (Genetics), Ultraviolet Rays}, issn = {0950-382X}, author = {Sandler, S J and McCool, J D and Do, T T and Johansen, R U} } @article {789, title = {Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.}, journal = {Mol Biochem Parasitol}, volume = {108}, year = {2000}, month = {2000 May}, pages = {187-97}, abstract = {Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2\% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95\% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.}, keywords = {Animals, Cryptosporidiosis, Cryptosporidium parvum, Genome, Protozoan, Genotype, Germ-Free Life, Humans, Mice, Mice, Knockout, Molecular Sequence Data, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Sequence Analysis, DNA, Swine}, issn = {0166-6851}, author = {Widmer, G and Akiyoshi, D and Buckholt, M A and Feng, X and Rich, S M and Deary, K M and Bowman, C A and Xu, P and Wang, Y and Wang, X and Buck, G A and Tzipori, S} }