Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness.Flagellar function in this organism is critical for life cycle progression and pathogenesis,however the regulation of flagellar motility is not well understood.The flagellar axoneme produces a complex beat through the pre-cisely coordinated firing of many proteins,including multiple dynein motors.These motors are found in the inner arm and outer arm complexes.We are studying one of the inner arm dynein motors in the T.brucei flagellum:dynein-f.RNAi knockdown of genes for two components of dynein-f:DNAH10,theαheavy chain,and IC138,an intermediate chain,cause severe motility defects including immotility.To determine if motility defects result from structural disruption of the axoneme,we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy(TEM).Our analysis showed that inner arm dynein size,axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures.These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.

Identification of a sensor histidine kinase (BfcK) controlling biofilm formation in Clostridium acetobutylicum

Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.