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1.
J Mol Diagn ; 21(1): 38-48, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30577886

RESUMO

Clinical exome sequencing (CES) has a reported diagnostic yield of 20% to 30% for most clinical indications. The ongoing discovery of novel gene-disease and variant-disease associations are expected to increase the diagnostic yield of CES. Performing systematic reanalysis of previously nondiagnostic CES samples represents a significant challenge for clinical laboratories. Here, we present the results of a novel automated reanalysis methodology applied to 300 CES samples initially analyzed between June 2014 and September 2016. Application of our reanalysis methodology reduced reanalysis variant analysis burden by >93% and correctly captured 70 of 70 previously identified diagnostic variants among 60 samples with previously identified diagnoses. Notably, reanalysis of 240 initially nondiagnostic samples using information available on July 1, 2017, revealed 38 novel diagnoses, representing a 15.8% increase in diagnostic yield. Modeling monthly iterative reanalysis of 240 nondiagnostic samples revealed a diagnostic rate of 0.57% of samples per month. Modeling the workload required for monthly iterative reanalysis of nondiagnostic samples revealed a variant analysis burden of approximately 5 variants/month for proband-only and approximately 0.5 variants/month for trio samples. Approximately 45% of samples required evaluation during each monthly interval, and 61.3% of samples were reevaluated across three consecutive reanalyses. In sum, automated reanalysis methods can facilitate efficient reevaluation of nondiagnostic samples using up-to-date literature and can provide significant value to clinical laboratories.

2.
MBio ; 8(6)2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29184015

RESUMO

It is well known that many bacteria can survive in a growth-arrested state for long periods of time, on the order of months or even years, without forming dormant structures like spores or cysts. How is such longevity possible? What is the molecular basis of such longevity? Here we used the Gram-negative phototrophic alphaproteobacterium Rhodopseudomonas palustris to identify molecular determinants of bacterial longevity. R. palustris maintained viability for over a month after growth arrest due to nutrient depletion when it was provided with light as a source of energy. In transposon sequencing (Tn-seq) experiments, we identified 117 genes that were required for long-term viability of nongrowing R. palustris cells. Genes in this longevity gene set are annotated to play roles in a number of cellular processes, including DNA repair, tRNA modification, and the fidelity of protein synthesis. These genes are critically important only when cells are not growing. Three genes annotated to affect translation or posttranslational modifications were validated as bona fide longevity genes by mutagenesis and complementation experiments. These genes and others in the longevity gene set are broadly conserved in bacteria. This raises the possibility that it will be possible to define a core set of longevity genes common to many bacterial species.IMPORTANCE Bacteria in nature and during infections often exist in a nongrowing quiescent state. However, it has been difficult to define experimentally the molecular characteristics of this crucial element of the bacterial life cycle because bacteria that are not growing tend to die under laboratory conditions. Here we present and validate the phototrophic bacterium Rhodopseudomonas palustris as a model system for identification of genes required for the longevity of nongrowing bacteria. Growth-arrested R. palustris maintained almost full viability for weeks using light as an energy source. Such cells were subjected to large-scale mutagenesis to identify genes required for this striking longevity trait. The results define conserved determinants of survival under nongrowing conditions and create a foundation for more extensive studies to elucidate general molecular mechanisms of bacterial longevity.


Assuntos
Viabilidade Microbiana , Rodopseudomonas/fisiologia , Elementos de DNA Transponíveis , Genes Bacterianos , Anotação de Sequência Molecular , Mutagênese Insercional , Rodopseudomonas/genética , Análise de Sequência de DNA
3.
J Bacteriol ; 198(5): 867-76, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26712940

RESUMO

UNLABELLED: Rhodopseudomonas palustris is an alphaproteobacterium that has served as a model organism for studies of photophosphorylation, regulation of nitrogen fixation, production of hydrogen as a biofuel, and anaerobic degradation of aromatic compounds. This bacterium is able to transition between anaerobic photoautotrophic growth, anaerobic photoheterotrophic growth, and aerobic heterotrophic growth. As a starting point to explore the genetic basis for the metabolic versatility of R. palustris, we used transposon mutagenesis and Tn-seq to identify 552 genes as essential for viability in cells growing aerobically on semirich medium. Of these, 323 have essential gene homologs in the alphaproteobacterium Caulobacter crescentus, and 187 have essential gene homologs in Escherichia coli. There were 24 R. palustris genes that were essential for viability under aerobic growth conditions that have low sequence identity but are likely to be functionally homologous to essential E. coli genes. As expected, certain functional categories of essential genes were highly conserved among the three organisms, including translation, ribosome structure and biogenesis, secretion, and lipid metabolism. R. palustris cells divide by budding in which a sessile cell gives rise to a motile swarmer cell. Conserved cell cycle genes required for this developmental process were essential in both C. crescentus and R. palustris. Our results suggest that despite vast differences in lifestyles, members of the alphaproteobacteria have a common set of essential genes that is specific to this group and distinct from that of gammaproteobacteria like E. coli. IMPORTANCE: Essential genes in bacteria and other organisms are those absolutely required for viability. Rhodopseudomonas palustris has served as a model organism for studies of anaerobic aromatic compound degradation, hydrogen gas production, nitrogen fixation, and photosynthesis. We used the technique of Tn-seq to determine the essential genes of R. palustris grown under heterotrophic aerobic conditions. The transposon library generated in this study will be useful for future studies to identify R. palustris genes essential for viability under specialized growth conditions and also for survival under conditions of stress.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Rodopseudomonas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , DNA Bacteriano , Biblioteca Gênica , Mutação
4.
J Bacteriol ; 195(7): 1525-37, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23354745

RESUMO

Previously, it was shown that an aconitase (citB) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing Bacillus subtilis cells, a phenotype that causes secondary effects, including the hyperexpression of the citB promoter. B. subtilis aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [citB2]) and the other designed to be defective in RNA binding (R741E [citB7]). The citB2 mutant was a glutamate auxotroph and accumulated citrate, while the citB7 mutant was a glutamate prototroph. Unexpectedly, the citB7 strain also accumulated citrate. Both mutant strains exhibited overexpression of the citB promoter and accumulated high levels of aconitase protein. These strains and the citB null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase (citZ) transcript was present at higher-than-normal levels in the citB null mutant, due at least in part to a >3-fold increase in the stability of the citZ transcript compared to the wild type. Purified B. subtilis aconitase bound to the citZ 5' leader RNA in vitro, but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the citZ transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.


Assuntos
Aconitato Hidratase/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ciclo do Ácido Cítrico/genética , Regulação Bacteriana da Expressão Gênica , Aconitato Hidratase/genética , Sítios de Ligação , Ácido Cítrico/metabolismo , DNA Bacteriano/metabolismo , Perfilação da Expressão Gênica , Ácido Glutâmico/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
5.
Microbiology ; 159(Pt 1): 68-76, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23139400

RESUMO

The role of the CcpC regulatory protein as a repressor of the genes encoding the tricarboxylic acid branch enzymes of the Krebs cycle (citrate synthase, citZ; aconitase, citB; and isocitrate dehydrogenase, citC) has been established for both Bacillus subtilis and Listeria monocytogenes. In addition, hyperexpression of citB-lacZ reporter constructs in an aconitase null mutant strain has been reported for B. subtilis. We show here that such hyperexpression of citB occurs in L. monocytogenes as well as in B. subtilis and that in both species the hyperexpression is unexpectedly dependent on CcpC. We propose a revision of the existing CcpC-citB regulatory scheme and suggest a mechanism of regulation in which CcpC represses citB expression at low citrate levels and activates citB expression when citrate levels are high.


Assuntos
Aconitato Hidratase/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/enzimologia , Listeria monocytogenes/genética , Proteínas Repressoras/metabolismo , Fusão Gênica Artificial , Deleção de Genes , Genes Reporter , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
6.
Proc Natl Acad Sci U S A ; 109(30): 12141-6, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22778415

RESUMO

Quorum sensing in the bacterium Rhodopseudomonas palustris involves the RpaI signal synthase, which produces p-coumaroyl-homoserine lactone (pC-HSL) and RpaR, which is a pC-HSL-dependent transcriptional activator. There is also an antisense rpaR transcript (asrpaR) of unknown function. Recent RNAseq studies have revealed that bacterial antisense RNAs are abundant, but little is known about the function of these molecules. Because asrpaR expression is quorum sensing dependent, we sought to characterize its production and function. We show that asrpaR is approximately 300-600 bases and is produced in response to pC-HSL and RpaR. There is an RpaR-binding site centered 51.5 bp from the mapped asrpaR transcript start site. We show that asrpaR overexpression reduces RpaR levels, rpaI expression, and pC-HSL production. We also generated an asrpaR mutant, which shows elevated RpaR levels, and elevated rpaI expression. Thus, asrpaR inhibits rpaR translation, and this inhibition results in suppression of RpaR-dependent rpaI expression and, thus, pC-HSL production. The R. palustris asrpaR represents an antisense RNA for which an activity can be measured and for which a distinct regulatory circuit related to a function is elucidated. It also represents yet another subtle regulatory layer for acyl-homoserine lactone quorum-sensing signal-responsive transcription factors.


Assuntos
Percepção de Quorum/genética , RNA Antissenso/metabolismo , Rodopseudomonas/genética , Rodopseudomonas/fisiologia , Transativadores/metabolismo , Acil-Butirolactonas/metabolismo , Sítios de Ligação/genética , Northern Blotting , Western Blotting , Primers do DNA/genética , Engenharia Genética/métodos , Mutagênese , Plasmídeos/genética , RNA Antissenso/genética , Transativadores/genética
7.
J Bacteriol ; 188(17): 6396-405, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16923907

RESUMO

Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of sigmaK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3' untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.


Assuntos
Aconitato Hidratase/fisiologia , Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas de Ligação a RNA/fisiologia , Aconitato Hidratase/genética , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação Puntual , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Esporos Bacterianos/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo
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