Biology subjects

Fiebig, A.

Publications and source records attributed to Fiebig, A..

5 recordsLinked to original sources

bioRxiv2018

A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly

Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. Caulobacter crescentus produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is a carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We have leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multi-day time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and flagellum control holdfast synthesis separately by modulating the holdfast inhibitor hfiA. This study defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of C. crescentus to surfaces.\n\nImportanceBacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. Caulobacter crescentus produces a structure called the holdfast, which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.

microbiology↗

bioRxiv2018

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded {beta}-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the integrity of the cell envelope. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

microbiology↗

bioRxiv2018

Evaluation of Caulobacter crescentus strain fitness at the genomic scale during growth in natural freshwater

Bacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacterium Caulobacter crescentus grown in natural freshwater, compared to axenic growth in common laboratory media. Gene disruptions in amino acid and nucleotide biosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting that Caulobacter does not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable in lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions between Caulobacter and a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

microbiology↗

bioRxiv2018

Molecular control of gene expression by Brucella BaaR, an IclR-family repressor

The Brucella abortus general stress response sigma factor, {sigma}E1, directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection. Characterizing the functions of {sigma}E1 regulated genes therefore contributes to understanding of B. abortus physiology and infection biology. Transcription of the IclR family regulator, Bab2_0215, is indirectly activated by {sigma}E1 but its function remains undefined. We present a structural and functional characterization of Bab2_0215, which we have named Brucella adipic acid activated regulator (BaaR). BaaR adopts a classic IclR-family fold and directly regulates the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and represses transcription. We identified three carboxylic acids (adipic acid tetradecanedioic acid, {varepsilon}-aminocaproic acid) and a lactone ({varepsilon}-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhance transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid requires the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a set of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program regulated downstream of {sigma}E1.

biochemistry↗

bioRxiv2017

Gene network analysis identifies a central post-transcriptional regulator of cellular stress survival

Cells adapt to shifts in their environment by remodeling transcription. Measuring changes in transcription at the genome scale is now routine, but defining the functional significance of individual genes within large gene expression datasets remains a major challenge. We applied a network-based algorithm to interrogate publicly available transcription data to predict genes that serve major functional roles in Caulobacter crescentus stress survival. This approach identified GsrN, a conserved small RNA that is directly controlled by the general stress sigma factor, {sigma}{tau}, and functions as a potent post-transcriptional regulator of survival under multiple stress conditions. GsrN expression is both necessary and sufficient to protect cells from hydrogen peroxide, where it functions by base pairing with the leader of katG mRNA and promoting catalase/peroxidase expression. We conclude that GsrN convenes a post-transcriptional layer of gene expression that serves a central functional role in stress physiology.

microbiology↗