Establishment Limitation Constrains the Abundance of Lactic Acid Bacteria in the Napa Cabbage Phyllosphere.

Patterns of phyllosphere diversity have become increasingly clear with high-throughput sequencing surveys, but the processes that control phyllosphere diversity are still emerging. Through a combination of lab and field experiments using Napa cabbage and lactic acid bacteria (LAB), we examined how dispersal and establishment processes shape the ecological distributions of phyllosphere bacteria. We first determined the abundance and diversity of LAB on Napa cabbage grown at three sites using both culture-based approaches and 16S rRNA gene amplicon sequencing. Across all sites, LAB made up less than 0.9% of the total bacterial community abundance. To assess whether LAB were low in abundance in the Napa cabbage phyllosphere due to a limited abundance in local species pools (source limitation), we quantified LAB in leaf and soil samples across 51 vegetable farms and gardens throughout the northeastern United States. Across all sites, LAB comprised less than 3.2% of the soil bacterial communities and less than 1.6% of phyllosphere bacterial communities. To assess whether LAB are unable to grow in the phyllosphere even if they dispersed at high rates (establishment limitation), we used a gnotobiotic Napa cabbage system in the lab with experimental communities mimicking various dispersal rates of LAB. Even at high dispersal rates, LAB became rare or completely undetectable in experimental communities, suggesting that they are also establishment limited. Collectively, our data demonstrate that the low abundance of LAB in phyllosphere communities may be explained by establishment limitation.IMPORTANCE The quality and safety of vegetable fermentations are dependent on the activities of LAB naturally present in the phyllosphere. Despite their critical role in determining the success of fermentation, the processes that determine the abundance and diversity of LAB in vegetables used for fermentation are poorly characterized. Our work demonstrates that the limited ability of LAB to grow in the cabbage phyllosphere environment may constrain their abundance on cabbage leaves. These results suggest that commercial fermentation of Napa cabbage proceeds despite low and variable abundances of LAB across different growing regions. Propagule limitation may also explain ecological distributions of other rare members of phyllosphere microbes.

Bacterial-fungal interactions promote parallel evolution of global transcriptional regulators in a widespread Staphylococcus species.

Experimental studies of microbial evolution have largely focused on monocultures of model organisms, but most microbes live in communities where interactions with other species may impact rates and modes of evolution. Using the cheese rind model microbial community, we determined how species interactions shape the evolution of the widespread food- and animal-associated bacterium Staphylococcus xylosus. We evolved S. xylosus for 450 generations alone or in co-culture with one of three microbes: the yeast Debaryomyces hansenii, the bacterium Brevibacterium aurantiacum, and the mold Penicillium solitum. We used the frequency of colony morphology mutants (pigment and colony texture phenotypes) and whole-genome sequencing of isolates to quantify phenotypic and genomic evolution. The yeast D. hansenii strongly promoted diversification of S. xylosus. By the end of the experiment, all populations co-cultured with the yeast were dominated by pigment and colony morphology mutant phenotypes. Populations of S. xylosus grown alone, with B. aurantiacum, or with P. solitum did not evolve novel phenotypic diversity. Whole-genome sequencing of individual mutant isolates across all four treatments identified numerous unique mutations in the operons for the SigB, Agr, and WalRK global regulators, but only in the D. hansenii treatment. Phenotyping and RNA-seq experiments highlighted altered pigment and biofilm production, spreading, stress tolerance, and metabolism of S. xylosus mutants. Fitness experiments revealed antagonistic pleiotropy, where beneficial mutations that evolved in the presence of the yeast had strong negative fitness effects in other biotic environments. This work demonstrates that bacterial-fungal interactions can have long-term evolutionary consequences within multispecies microbiomes by facilitating the evolution of strain diversity.

On an unbiased and consistent estimator for mutation rates.

Spontaneous mutations are stochastic events. The mutation rate, defined as mutations per genome per replication, is generally very low, and it is widely accepted that spontaneous mutations occur at defined, but different, rates in bacteriophage and in bacterial, insect, and mammalian cells. The calculation of mutation rates has proved to be a significant problem. Mutation rates can be calculated by following mutant accumulation during growth or from the distribution of mutants obtained in parallel cultures. As Luria and Delbrück described in 1943, the number of mutants in parallel populations of bacterial cells varies widely depending on when a spontaneous mutation occurs during growth of the culture. Since 1943, many mathematical refinements to estimating rates, called estimators, have been described to facilitate determination of the mutation rate from the distribution or frequency of mutants detected following growth of parallel cultures. We present a rigorous mathematical solution to the mutation rate problem using an unbiased and consistent estimator. Using this estimator we demonstrate experimentally that mutation rates can be easily calculated by determining mutant accumulation, that is, from the number of mutants measured in two successive generations. Moreover, to verify the consistency of our estimator we conduct a series of simulation trials that show a surprisingly rapid convergence to the targeted mutation rate (reached between 25th and 30th generations).

DNA Replication-Transcription Conflicts Do Not Significantly Contribute to Spontaneous Mutations Due to Replication Errors in Escherichia coli.

Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch repair (MMR)-defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base pair substitutions or small indels (i.e., insertion and deletions of ≤4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hot spots for indels. No additional orientation bias in mutation frequencies occurred when MMR- strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus codirectional to replication. In addition, the data revealed a strong correlation of the frequency of base pair substitutions with gene length but no correlation with gene expression levels. IMPORTANCE Because DNA replication and transcription occur on the same DNA template, encounters between the two machines occur frequently. When these encounters are head-to-head, genomic disruption can occur. However, whether replication-transcription conflicts contribute to spontaneous mutations is debated. Analyzing in detail a large collection of mutations generated with mismatch repair-defective Escherichia coli strains, we found that across the genome there are no significant differences in mutation frequencies between genes oriented codirectionally and those oriented head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for small insertions and deletions in head-on-oriented genes, but this difference was almost entirely due to the asymmetrical locations of tRNA genes containing mononucleotide runs, which are hot spots for these mutations. Thus, biases in the positions of mutational target sequences can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on and codirectionally to replication.

Strain-Level Diversity Impacts Cheese Rind Microbiome Assembly and Function.

Diversification can generate genomic and phenotypic strain-level diversity within microbial species. This microdiversity is widely recognized in populations, but the community-level consequences of microbial strain-level diversity are poorly characterized. Using the cheese rind model system, we tested whether strain diversity across microbiomes from distinct geographic regions impacts assembly dynamics and functional outputs. We first isolated the same three bacterial species (Staphylococcus equorum, Brevibacterium auranticum, and Brachybacterium alimentarium) from nine cheeses produced in different regions of the United States and Europe to construct nine synthetic microbial communities consisting of distinct strains of the same three bacterial species. Comparative genomics identified distinct phylogenetic clusters and significant variation in genome content across the nine synthetic communities. When we assembled each synthetic community with initially identical compositions, community structure diverged over time, resulting in communities with different dominant taxa. The taxonomically identical communities showed differing responses to abiotic (high salt) and biotic (the fungus Penicillium) perturbations, with some communities showing no response and others substantially shifting in composition. Functional differences were also observed across the nine communities, with significant variation in pigment production (light yellow to orange) and in composition of volatile organic compound profiles emitted from the rinds (nutty to sulfury).IMPORTANCE Our work demonstrated that the specific microbial strains used to construct a microbiome could impact the species composition, perturbation responses, and functional outputs of that system. These findings suggest that 16S rRNA gene taxonomic profiles alone may have limited potential to predict the dynamics of microbial communities because they usually do not capture strain-level diversity. Observations from our synthetic communities also suggest that strain-level diversity has the potential to drive variability in the aesthetics and quality of surface-ripened cheeses.

New complexities of SOS-induced "untargeted" mutagenesis in Escherichia coli as revealed by mutation accumulation and whole-genome sequencing.

When its DNA is damaged, Escherichia coli induces the SOS response, which consists of about 40 genes that encode activities to repair or tolerate the damage. Certain alleles of the major SOS-control genes, recA and lexA, cause constitutive expression of the response, resulting in an increase in spontaneous mutations. These mutations, historically called "untargeted", have been the subject of many previous studies. Here we re-examine SOS-induced mutagenesis using mutation accumulation followed by whole-genome sequencing (MA/WGS), which allows a detailed picture of the types of mutations induced as well as their sequence-specificity. Our results confirm previous findings that SOS expression specifically induces transversion base-pair substitutions, with rates averaging about 60-fold above wild-type levels. Surprisingly, the rates of G:C to C:G transversions, normally an extremely rare mutation, were induced an average of 160-fold above wild-type levels. The SOS-induced transversion showed strong sequence specificity, the most extreme of which was the G:C to C:G transversions, 60% of which occurred at the middle base of 5'GGC3'+5'GCC3' sites, although these sites represent only 8% of the G:C base pairs in the genome. SOS-induced transversions were also DNA strand-biased, occurring, on average, 2- to 4- times more often when the purine was on the leading-strand template and the pyrimidine on the lagging-strand template than in the opposite orientation. However, the strand bias was also sequence specific, and even of reverse orientation at some sites. By eliminating constraints on the mutations that can be recovered, the MA/WGS protocol revealed new complexities of SOS "untargeted" mutations.

The Symmetrical Wave Pattern of Base-Pair Substitution Rates across the Escherichia coli Chromosome Has Multiple Causes.

Mutation accumulation experiments followed by whole-genome sequencing have revealed that, for several bacterial species, the rate of base-pair substitutions (BPSs) is not constant across the chromosome but varies in a wave-like pattern that is symmetrical about the origin of replication. The experiments reported here demonstrated that, in Escherichia coli, several interacting factors determine the wave. The origin is a major driver of BPS rates. When it is relocated, the BPS rates in a 1,000-kb region surrounding the new origin reproduce the pattern that surrounds the normal origin. However, the pattern across distant regions of the chromosome is unaltered and thus must be determined by other factors. Increasing the deoxynucleoside triphosphate (dNTP) concentration shifts the wave pattern away from the origin, supporting the hypothesis that fluctuations in dNTP pools coincident with replication firing contribute to the variations in the mutation rate. The nucleoid binding proteins (HU and Fis) and the terminus organizing protein (MatP) are also major factors. These proteins alter the three-dimensional structure of the DNA, and results suggest that mutation rates increase when highly structured DNA is replicated. Biases in error correction by proofreading and mismatch repair, both of which may be responsive to dNTP concentrations and DNA structure, also are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.IMPORTANCE It has been found in several species of bacteria that the rate at which single base pairs are mutated is not constant across the genome but varies in a wave-like pattern that is symmetrical about the origin of replication. Using Escherichia coli as our model system, we show that this pattern is the result of several interconnected factors. First, the timing and progression of replication are important in determining the wave pattern. Second, the three-dimensional structure of the DNA is also a factor, and the results suggest that mutation rates increase when highly structured DNA is replicated. Finally, biases in error correction, which may be responsive both to the progression of DNA synthesis and to DNA structure, are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.

Role of Hfq in Genome Evolution: Instability of G-Quadruplex Sequences in E. coli.

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli.

When the DNA polymerase that replicates the Escherichia coli chromosome, DNA polymerase III, makes an error, there are two primary defenses against mutation: proofreading by the ϵ subunit of the holoenzyme and mismatch repair. In proofreading-deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole-genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli's low mutation rate of 1 per 1000 generations.

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair Defective Escherichia coli.

Mismatch repair (MMR) is a major contributor to replication fidelity, but its impact varies with sequence context and the nature of the mismatch. Mutation accumulation experiments followed by whole-genome sequencing of MMR-defective Escherichia coli strains yielded ≈30,000 base-pair substitutions (BPSs), revealing mutational patterns across the entire chromosome. The BPS spectrum was dominated by A:T to G:C transitions, which occurred predominantly at the center base of 5'NAC3'+5'GTN3' triplets. Surprisingly, growth on minimal medium or at low temperature attenuated these mutations. Mononucleotide runs were also hotspots for BPSs, and the rate at which these occurred increased with run length. Comparison with ≈2000 BPSs accumulated in MMR-proficient strains revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be sites of frequent replication errors. In MMR-defective strains transitions were strand biased, occurring twice as often when A and C rather than T and G were on the lagging-strand template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In an mmr ndk double mutant strain, these mutations were more frequent when the template A and T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone, or less well corrected by proofreading, than was leading strand synthesis.