To study how a bacterium allocates its resources, we compared the costs and benefits of most (86%) of the proteins in Escherichia coli K-12 during growth in minimal glucose medium. The cost or investment in each protein was estimated from ribosomal profiling data, and the benefit of each protein was measured by assaying a library of transposon mutants. We found that proteins that are important for fitness are usually highly expressed, and 95% of these proteins are expressed at above 13 parts per million (ppm). Conversely, proteins that do not measurably benefit the host (with a benefit of less than 5% per generation) tend to be weakly expressed, with a median expression of 13 ppm. In aggregate, genes with no detectable benefit account for 31% of protein production, or about 22% if we correct for genetic redundancy. Although some of the apparently unnecessary expression could have subtle benefits in minimal glucose medium, the majority of the burden is due to genes that are important in other conditions. We propose that at least 13% of the cell's protein is "on standby" in case conditions change.

To study how a bacterium allocates its resources, we compared the costs and benefits of most of the proteins in Escherichia coli K-12 during growth in minimal glucose medium. Proteins that are important for fitness are usually highly expressed, and 95% of these proteins are expressed at above 13 parts per million (ppm). Conversely, proteins that do not measurably benefit the host tend to be weakly expressed, with a median expression of 13 ppm. In aggregate, genes with no detectable benefit account for 31% of protein production, or about 22% if we correct for genetic redundancy. Although some of the apparently unnecessary expression could have subtle benefits in minimal glucose medium, the majority of the burden is due to genes that are important in other conditions. We propose that over 10% of the cell’s protein is “on standby” in case conditions change.

The fitness effects of all possible mutations available to an organism largely shape the dynamics of evolutionary adaptation. Yet, whether and how this adaptive landscape changes over evolutionary times, especially upon ecological diversification and changes in community composition, remains poorly understood. We sought to fill this gap by analyzing a stable community of two closely related ecotypes ("L" and "S") shortly after they emerged within the E. coli Long-Term Evolution Experiment (LTEE). We engineered genome-wide barcoded transposon libraries to measure the invasion fitness effects of all possible gene knockouts in the coexisting strains as well as their ancestor, for many different, ecologically relevant conditions. We find consistent statistical patterns of fitness effect variation across both genetic background and community composition, despite the idiosyncratic behavior of individual knockouts. Additionally, fitness effects are correlated with evolutionary outcomes for a number of conditions, possibly revealing shifting patterns of adaptation. Together, our results reveal how ecological and epistatic effects combine to shape the adaptive landscape in a nascent ecological community.

Magnetotactic bacteria (MTB) are a phylogenetically diverse group of bacteria remarkable for their ability to biomineralize magnetite (Fe₃O₄) or greigite (Fe₃S₄) in organelles called magnetosomes. The majority of genes required for magnetosome formation are encoded by a magnetosome gene island (MAI). Most previous genetic studies of MTB have focused on the MAI, using screens to identify key MAI genes or targeted genetics to isolate specific genes and their function in one specific growth condition. This is the first study that has taken an unbiased approach to look at many different growth conditions to reveal key genes both inside and outside the MAI. Here, we conducted random barcoded transposon mutagenesis (RB-TnSeq) in Magnetospirillum magneticum AMB-1. We generated a library of 184,710 unique strains in a wild-type background, generating ∼34 mutant strains for each gene. RB-TnSeq also allowed us to determine the essential gene set of AMB-1 under standard laboratory growth conditions. To pinpoint novel genes that are important for magnetosome formation, we subjected the library to magnetic selection screens under varied growth conditions. We compared biomineralization under standard growth conditions to biomineralization under high-iron and anaerobic conditions, respectively. Strains with transposon insertions in the MAI gene mamT had an exacerbated biomineralization defect under both high-iron and anaerobic conditions compared to standard conditions, adding to our knowledge of the role of MamT in magnetosome formation. Mutants in an ex-MAI gene, amb4151, are more magnetic than wild-type cells under anaerobic conditions. All three of these phenotypes were validated by creating a markerless deletion strain of the gene and evaluating with TEM imaging. Overall, our results indicate that growth conditions affect which genes are required for biomineralization and that some MAI genes may have more nuanced functions than was previously understood. IMPORTANCE Magnetotactic bacteria (MTB) are a group of bacteria that can form nano-sized crystals of magnetic minerals. MTB are likely an important part of their ecosystems, because they can account for up to a third of the microbial biomass in an aquatic habitat and consume large amounts of iron, potentially impacting the iron cycle. The ecology of MTB is relatively understudied; however, the cell biology and genetics of MTB have been studied for decades. Here, we leverage genetic studies of MTB to inform environmental studies. We expand the genetic toolset for studying MTB in the lab and identify novel genes, or functions of genes, that have an impact on biomineralization.

We performed whole genome analyses of DNA methylation in Shewanella17 oneidensis MR-1 to examine its possible role in regulating gene expression and18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing19 revealed extensive methylation of adenine (N6mA) throughout the20 genome. These methylated bases were located in five sequence motifs,21 including three novel targets for Type I restriction/modification enzymes. The22 sequence motifs targeted by putative methyltranferases were determined via23 SMRT sequencing of gene knockout mutants. In addition, we found S.24 oneidensis MR-1 cultures grown under various culture conditions displayed25 different DNA methylation patterns. However, the small number of differentially26 methylated sites could not be directly linked to the much larger number of27 differentially expressed genes in these conditions, suggesting DNA methylation is28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment29 of methylated GATC motifs in the origin of replication indicate DNA methylation30 may regulate genome replication in a manner similar to that seen in Escherichia31 coli. Furthermore, comparative analyses suggest that many32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.

Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguously identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity and pathogen resistance.

Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.

Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.

Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13) is sometimes important alongside Rnf and may perform an electron bifurcation to generate more reduced ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1) is important and may generate reduced ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions, although we cannot rule out genetic redundancy.

Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes' function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory. In Shewanella oneidensis MR-1, 24% of genes are detrimental to fitness in some conditions, and detrimental genes tend to be highly expressed instead of being repressed when not needed. In diverse bacteria, there is little correlation between when genes are important for optimal growth or fitness and when those genes are upregulated. Two common types of indirect control are constitutive expression and regulation by growth rate; these occur for genes with diverse functions and often seem to be suboptimal. Because genes that have closely related functions can have dissimilar expression patterns, regulation may be suboptimal in the wild as well as in the laboratory.