Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion

Abstract Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40°C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.     

Alpha-complementation as a probe for dual localization of mitochondrial proteins

There are a growing number of proteins which are reported to reside in multiple compartments within the eukaryotic cell. However, lack of appropriate methods limits our knowledge on the true extent of this phenomenon. In this study, we demonstrate a novel application of beta-galactosidase alpha-complementation to study dual distribution of proteins in yeast cells. Using a simple colony color phenotype, we show that alpha-complementation depends on co-compartmentalization of alpha and omega fragments and exploit this to probe dual localization of proteins between the cytosol and mitochondria in yeast. The quality of our assay was assessed by analysis of the known dual targeted enzyme fumarase and several mutant derivatives, which are exclusively localized to one or the other of these subcellular compartments. Addition of the alpha fragment did not abolish the enzymatic activity of the tagged proteins nor did it affect their localization. By examining 10 yeast gene products for distribution between the cytosol and the mitochondria, we demonstrate the potential of alpha-complementation to screen the mitochondrial proteome for dual distribution. Our data indicate the distribution of two uncharacterized proteins--Bna3 and Nif3--between the cytosol and the mitochondria.     

Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli

Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.     

Systematic Identification of Rhythmic Genes Reveals camk1gb as a New Element in the Circadian Clockwork

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.     

Exploring the bovine rumen bacterial community from birth to adulthood

The mammalian gut microbiota is essential in shaping many of its host''s functional attributes. One such microbiota resides in the bovine digestive tract in a compartment termed as the rumen. The rumen microbiota is necessary for the proper physiological development of the rumen and for the animal''s ability to digest and convert plant mass into food products, making it highly significant to humans. The establishment of this microbial population and the changes occurring with the host''s age are important for understanding this key microbial community. Despite its importance, little information about colonization of the microbial populations in newborn animals, and the gradual changes occurring thereafter, exists. Here, we characterized the overall bovine ruminal bacterial populations of five age groups, from 1-day-old calves to 2-year-old cows. We describe the changes occurring in the rumen ecosystem after birth, reflected by a decline in aerobic and facultative anaerobic taxa and an increase in anaerobic ones. Some rumen bacteria that are essential for mature rumen function could be detected as early as 1 day after birth, long before the rumen is active or even before ingestion of plant material occurs. The diversity and within-group similarity increased with age, suggesting a more diverse but homogeneous and specific mature community, compared with the more heterogeneous and less diverse primary community. In addition, a convergence toward a mature bacterial arrangement with age was observed. These findings have also been reported for human gut microbiota, suggesting that similar forces drive the establishment of gut microbiotas in these two distinct mammalian digestive systems.