Single Cells Exhibit Differing Behavioral Phases during Early Stages of Pseudomonas aeruginosa Swarming.

Pseudomonas aeruginosa is among the many bacteria that swarm, where groups of cells coordinate to move over surfaces. It has been challenging to determine the behavior of single cells within these high-cell-density swarms. To track individual cells within P. aeruginosa swarms, we imaged a fluorescently labeled subset of the larger population. Single cells at the advancing swarm edge varied in their motility dynamics as a function of time. From these data, we delineated four phases of early swarming prior to the formation of the tendril fractals characteristic of P. aeruginosa swarming by collectively considering both micro- and macroscale data. We determined that the period of greatest single-cell motility does not coincide with the period of greatest collective swarm expansion. We also noted that flagellar, rhamnolipid, and type IV pilus motility mutants exhibit substantially less single-cell motility than the wild type.IMPORTANCE Numerous bacteria exhibit coordinated swarming motion over surfaces. It is often challenging to assess the behavior of single cells within swarming communities due to the limitations of identifying, tracking, and analyzing the traits of swarming cells over time. Here, we show that the behavior of Pseudomonas aeruginosa swarming cells can vary substantially in the earliest phases of swarming. This is important to establish that dynamic behaviors should not be assumed to be constant over long periods when predicting and simulating the actions of swarming bacteria.

Assessing Travel Conditions: Environmental and Host Influences On Bacterial Surface Motility.

The degree to which surface motile bacteria explore their surroundings is influenced by aspects of their local environment. Accordingly, regulation of surface motility is controlled by numerous chemical, physical, and biological stimuli. Discernment of such regulation due to these multiple cues is a formidable challenge. Additionally inherent ambiguity and variability from the assays used to assess surface motility can be an obstacle to clear delineation of regulated surface motility behavior. Numerous studies have reported single environmental determinants of microbial motility and lifestyle behavior but the translation of these data to understand surface motility and bacterial colonization of human host or environmental surfaces is unclear. Here, we describe the current state of the field and our understanding of exogenous factors that influence bacterial surface motility.

Selenite activates the alternative oxidase pathway and alters primary metabolism in Brassica napus roots: evidence of a mitochondrial stress response.

BACKGROUND: Human requirements for dietary selenium are met mainly by crops. However, excessive uptake of selenium in plants can restrict growth, and its toxicity has been postulated to target roots. Selenite toxicity can be attributed to its assimilation into selenocysteine, which can replace cysteine to yield malformed selenoproteins. Additionally, selenite has pro-oxidant properties. In this study, the effects of selenite on root tissue in Brassica napus (canola) were investigated to better understand its mode of toxicity and the metabolic adjustments needed to mediate a selenite-response. RESULTS: Selenite induced the rapid formation of mitochondrial superoxide, which led to decreased aconitase activity and involvement of the alternative oxidase pathway. Although selenite altered primary metabolism, as observed by the increased amino acids and decreased TCA cycle metabolites, increased glucose presumably supported higher respiratory rates and ATP levels reported in this study. Additionally, evidence is presented indicating that selenite suppressed the ubiquitin-proteasome pathway, and induced the pentose phosphate pathway needed to maintain antioxidant metabolism. Selenite treatment also elevated glutathione concentration and coincided with increased levels of γ-glutamyl cyclotransferase, which may possibly degrade selenium metabolites conjugated to glutathione. CONCLUSION: Collectively, the data indicate that selenite necessitates the reconfiguration of metabolic pathways to overcome the consequences of mitochondrial oxidative stress in root tissue. Efforts to mitigate the detrimental effects of selenite-induced oxidative stress may ultimately improve selenium tolerance and accumulation in crops.

Characterization of tRNALeu binding interactions with Cu2+ and Pb2+ and their biological implications.

RNA is known to interact with Mg2+ when assuming higher-ordered tertiary configurations. Structurally, when tRNA molecules interact with Mg2+, they consistently form a "L-shape" conformation each time they are synthesized. Therefore, if Mg2+ can induce tertiary structure formation, then binding to alternative cations could produce alternative tertiary configurations. By utilizing circular dichroism and mobility gel-shift assays it was observed that tRNA structure can be altered when in the presence of different divalent cationic species. Formation of these alternative structural configurations was further validated by aminoacylating these tRNA structural anomalies with their native enzyme, which resulted in markedly different degrees of activity. Thus, it was confirmed that structural changes do occur when tRNA forms complexes with different cations. To better understand these structural changes, quantitative cation binding to tRNA was determined through titrations as well as ICP-OES analysis, which indicated that the metal ions can bind to the tRNA structure in specific and non-specific ways. Lastly, it was observed through stopped-flow kinetics that tRNA can associate/dissociate from different cations to varying degrees, thus forming cation-specific complexes at unique rates.

Stuck between a ROS and a hard place: Analysis of the ubiquitin proteasome pathway in selenocysteine treated Brassica napus reveals different toxicities during selenium assimilation.

During the selenium assimilation pathway, inorganic selenate and selenite are reduced to form selenocysteine (Sec). Tolerance to selenium in plants has long been attributable to minimizing the replacement of cysteine with selenocysteine, which can result in nonspecific selenoproteins that are potentially misfolded. Despite this widely accepted assumption, there is no evidence in higher plants demonstrating that selenocysteine induces toxicity by resulting in malformed proteins. In this study, we use Brassica napus to analyze the ubiquitin-proteasome pathway, which is capable of removing misfolded proteins. Sec rapidly increased proteasome activity and levels of ubiquitinated proteins, strongly indicating that selenocysteine induces protein misfolding. Proteasome inhibition increased the amount of selenium in protein in Sec-treated plants. Collectively, these data provide a mechanism that accounts for Sec toxicity. Additionally, Sec did not cause oxidative stress as judged by examining levels of superoxide using fluorescent microscopy. Therefore, the cellular response to Sec is different compared to selenite, which was recently shown to increase antioxidant metabolism in response to elevated mitochondrial superoxide that ultimately impaired proteasome activity. Therefore, plants must contend with two divergent modes of cytotoxicity during selenium assimilation. Selenite can result in oxidative stress, but increased flux of selenite reduction can yield Sec that in turn can cause protein misfolding.

Zinc is the molecular "switch" that controls the catalytic cycle of bacterial leucyl-tRNA synthetase.

The Escherichia coli (E. coli) leucyl-tRNA synthetase (LeuRS) enzyme is part of the aminoacyl-tRNA synthetase (aaRS) family. LeuRS is an essential enzyme that relies on specialized domains to facilitate the aminoacylation reaction. Herein, we have biochemically characterized a specialized zinc-binding domain 1 (ZN-1). We demonstrate that the ZN-1 domain plays a central role in the catalytic cycle of E. coli LeuRS. The ZN-1 domain, when associated with Zn(2+), assumes a rigid architecture that is stabilized by thiol groups from the residues C159, C176 and C179. When LeuRS is in the aminoacylation complex, these cysteine residues form an equilateral planar triangular configuration with Zn(2+), but when LeuRS transitions to the editing conformation, this geometric configuration breaks down. By generating a homology model of LeuRS while in the editing conformation, we conclude that structural changes within the ZN-1 domain play a central role in LeuRS's catalytic cycle. Additionally, we have biochemically shown that C159, C176 and C179 coordinate Zn(2+) and that this interaction is essential for leucylation to occur, but is not essential for deacylation. Furthermore, calculated Kd values indicate that the wild-type enzyme binds Zn(2+) to a greater extent than any of the mutant LeuRSs. Lastly, we have shown through secondary structural analysis of our LeuRS enzymes that Zn(2+) is an architectural cornerstone of the ZN-1 domain and that without its geometric coordination the domain collapses. We believe that future research on the ZN-1 domain may reveal a possible Zn(2+) dependent translocation mechanism for charged tRNA(Leu).