Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater

Abstract

25Bacterial genomes are the products of selection in fluctuating, spatially heterogeneous, and often 26 nutrient-poor environments. Thus bacterial gene function is best understood in this natural context, but 27 replicating such conditions in the lab is challenging. Here we used a transposon sequencing approach 28 to globally measure the fitness consequences of gene disruption in the free-living freshwater bacterium 29Caulobacter crescentus, cultivated in complex peptone broth, a defined mineral medium (M2-xylose), 30 or in littoral waters of Lake Michigan. Transposon insertions in genes involved in amino acid and 31 nucleotide biosynthesis and in transport resulted in large fitness defects in both lake water and M2-32 xylose relative to a complex peptone broth, though defects were more extreme in M2-xylose in most 33 cases. In lake water, strain fitness was increased by insertions in genes required for flagellum 34 biosynthesis and reduced by insertions in genes involved in holdfast biosynthesis. We further 35 uncovered numerous hypothetical and uncharacterized genes for which disruption resulted in reduced 36 fitness in either lake water, M2-xylose, or both. At the genomic scale, the fitness profile of the strain 37 pool cultivated in lake water was more similar to that in complex peptone broth than in M2-xylose. 38Individual gene and strain fitness scores were significantly more variable in lake water than in M2-39 xylose, presumably owing to variability in water conditions collected on different days. This study 40 advances our understanding of the genetic determinants of Caulobacter growth in a complex natural 41 environment, and provides a new avenue to study gene function in a free-living freshwater bacterium. 42 43 IMPORTANCE 44Bacteria live in complex environments that are variable over time and space. Bacterial genomes encode 45 the molecular machinery that allows the cell to interact with its environment and with other organisms. 46Much of this machinery remains uncharacterized, in part because it is difficult to simulate natural 47 conditions in the laboratory. Using the model bacterium Caulobacter crescentus, we developed an 48.

Role of Caulobacter Cell Surface Structures in Colonization of the Air-Liquid Interface

“…To visualize and monitor colonization of the air-liquid interface, I grew wild-type C. crescentus strain CB15 statically in large volumes of a peptone-yeast extract (PYE) broth. A recent genome-scale analysis of C. crescentus indicates that complex media, such as PYE, are a more ecologically relevant cultivation environment than a mineral defined medium, such as M2 (34). Under these conditions, as culture density increased, cells formed a surface film, or pellicle, that evenly covered the entire air-liquid interface (Fig.…”

“…I wondered whether C. crescentus could form a pellicle film in a more nutrient-limited environment, i.e., in a medium that did not support such high cell density. To address this question, I used a series of increasingly dilute complex media rather than a mineral defined M2-based medium for two main reasons: (i) standard M2-based media support dense cell growth (i.e., are not growth limiting) and (ii) there is evidence that complex media better reflect the complex suite of nutrients encountered by Caulobacter in natural ecosystems (34). While progressive dilution of a complex medium certainly reduced culture density (and cell size), a thick pellicle was observed in 0.1ϫ PYE, large rosettes accumulated at the surface of 0.03ϫ PYE, and small clusters of 2 to 4 cell rosettes formed on the surfaces of cultures grown in 0.01ϫ PYE (Fig.…”

Genome-wide identification of tomato xylem sap fitness factors for three plant-pathogenicRalstoniaspecies

“…In contrast, an RB-TnSeq study using Caulobacter crescentus shows that lake water, the natural environment of Caulobacter, is a dilute rich medium (36). Amino acid and vitamin auxotrophic Caulobacter mutants had less severe fitness defects in lake water than in minimal medium.…”

Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

“…Looking forward, we envision the ability to engineer self-synthesis and self-organization of a rationally-designed synthetic extracellular protein matrix will lead to autonomously assembled living materials. Such a material could be formed without intervention, induction chemicals, or applied force via an oligotroph that does not require complex nutrients due to its high synthetic capacity (33,64) , making it a uniquely low-cost, low-effort living material. These ELMs would also be modular based on the biopolymer design and cell count.…”

“…it is genetically tractable, is well-characterized due to its intriguing dimorphic life cycle (30) , strongly adheres to surfaces via its holdfast matrix (31) , and it has a modifiable proteinaceous Surface-layer (S-layer) (25,32) . In addition, it is oligotrophic and can flourish with minimal nutrients and in cold temperatures (33) . We previously reported the construction of a set of C.…”