RESUMO
Type IV pilus (T4P) dynamics is important for various bacterial functions including host cell interaction, surface motility, and horizontal gene transfer. T4P retract rapidly by depolymerization, generating large mechanical force. The gene that encodes the pilus retraction ATPase PilT has multiple paralogues, whose number varies between different bacterial species, but their role in regulating physical parameters of T4P dynamics remains unclear. Here, we address this question in the human pathogen Neisseria gonorrhoeae, which possesses two pilT paralogues, namely pilT2 and pilU. We show that the speed of twitching motility is strongly reduced in a pilT2 deletion mutant, while directional persistence time and sensitivity of speed to oxygen are unaffected. Using laser tweezers, we found that the speed of single T4P retraction was reduced by a factor of ≈ 2 in a pilT2 deletion strain, whereas pilU deletion showed a minor effect. The maximum force and the probability for switching from retraction to elongation under application of high force were not significantly affected. We conclude that the physical parameters of T4P are fine-tuned through PilT2.
Assuntos
Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Locomoção , Neisseria gonorrhoeae/fisiologia , Proteínas de Fímbrias/genética , Técnicas de Inativação de Genes , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismoAssuntos
Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Corantes Fluorescentes/química , Nanoestruturas/química , Fármacos Fotossensibilizantes/química , Zeolitas/química , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/química , Escherichia coli/metabolismo , Corantes Fluorescentes/farmacologia , Microscopia de Fluorescência , Fármacos Fotossensibilizantes/farmacologia , Coloração e Rotulagem , Zeolitas/farmacologiaRESUMO
Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus, T4P are unipolarly localized and undergo pole-to-pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non-piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole-to-pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP-PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.
Assuntos
Proteínas de Fímbrias/análise , Fímbrias Bacterianas/química , Proteínas Motores Moleculares/análise , Myxococcus xanthus/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Polaridade Celular/genética , Sequência Conservada/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Dados de Sequência Molecular , Movimento , Mutagênese Sítio-Dirigida , Mutação , Myxococcus xanthus/genética , Myxococcus xanthus/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The species human parvovirus B19 (B19V) can be divided into three genotypes. In this study, we addressed the question as to whether infection of an individual is restricted to one genotype. As viral DNA is detectable in tissue for long times after acute infection, we examined 87 liver specimens from adults for the presence of B19V DNA. Fifty-nine samples were found to be positive, 32 of them for genotype 1, 27 for genotype 2 and four for genotype 3. In four samples, DNA of two genotypes was detected; samples from three individuals were positive for genotypes 1 and 2 and a sample from one individual was positive for genotypes 1 and 3. Surprisingly, significant sequence heterogeneity was observed at approximately 1 % of the nucleotides of the genotype 1 genomes from individuals with double genotype 1 and 2 infection. Controls using different enzymes for genome amplification and dilutions of the template verified that nucleotide heterogeneity was due to the presence of three or more genome variants of genotype 1. In summary, the evidence shows that individuals can be infected with two different genotypes, and B19V DNA can persist as a population of different genomes. The results may have implications for the understanding of the antiviral immune response and the development of vaccines against B19V.
