In natural environments, bacteria survive conditions of starvation and stress. Long-term batch cultures are an excellent laboratory system to study adaptation during nutrient stress because cells can incubate for months to years without the addition of nutrients. During long-term batch culture, cells adapt to acquire energy from cellular detritus, creating a complex and dynamic environment for mutants of increased relative fitness to exploit. Here, we analyzed the genomes of 1,117 clones isolated from a single long-term batch culture incubated for 1,200 days. A total of 679 mutations included single nucleotide polymorphisms, indels, mobile genetic element movement, large deletions up to 64 kbp, and amplifications up to ∼500 kbp. During the 3.3-year incubation, two main lineages diverged, evolving continuously. At least twice, a previously fixed mutation reverted back to the wild-type allele, suggesting beneficial mutations may later become maladaptive due to the dynamic environment and changing selective pressures. Most of the mutated genes encode proteins involved in metabolism, transport, or transcriptional regulation. Clones from the two lineages are physiologically distinct, based on outgrowth in fresh medium and competition against the parental strain. Similar population dynamics and mutations in hfq, rpoS, paaX, lrp, sdhB, and dtpA were detected in three additional parallel populations sequenced through day 60, providing evidence for positive selection. These data provide new insight into the population structure and mutations that may be beneficial during periods of starvation in evolving bacterial communities. IMPORTANCE Bacteria have remarkable metabolic capabilities and adaptive plasticity, enabling them to survive in changing environments. In nature, bacteria spend a majority of their time in a state of slow growth or maintenance, scavenging nutrients for survival. Here, a population of Escherichia coli cells was incubated for 1,200 days in long-term batch culture, without the addition of new medium, requiring cells to continuously recycle nutrients. Whole-genome resequencing of cells from the evolving population identified two dominant subpopulations that coexisted while continuously acquiring and fixing new mutations. The population dynamics and alleles identified provide insight into adaptation to nutrient stress. Elucidating mechanisms that allow bacteria to adapt through cycles of feast and famine deepens our understanding of their survival mechanisms in nature.

ExoS/ChvI two-component signaling in the nitrogen-fixing α-proteobacterium Sinorhizobium meliloti is required for symbiosis and regulates exopolysaccharide production, motility, cell envelope integrity and nutrient utilization in free-living bacteria. However, identification of many ExoS/ChvI direct transcriptional target genes has remained elusive. Here, we performed chromatin immunoprecipitation followed by microarray analysis (chIP-chip) to globally identify DNA regions bound by ChvI protein in S. meliloti. We then performed qRT-PCR with chvI mutant strains to test ChvI-dependent expression of genes downstream of the ChvI-bound DNA regions. We identified 64 direct target genes of ChvI, including exoY, rem and chvI itself. We also identified ChvI direct target candidates, like exoR, that are likely controlled by additional regulators. Analysis of upstream sequences from the 64 ChvI direct target genes identified a 15 bp-long consensus sequence. Using electrophoretic mobility shift assays and transcriptional fusions with exoY, SMb21440, SMc00084, SMc01580, chvI, and ropB1, we demonstrated this consensus sequence is important for ChvI binding to DNA and transcription of ChvI direct target genes. Thus, we have comprehensively identified ChvI regulon genes and a 'ChvI box' bound by ChvI. Many ChvI direct target genes may influence the cell envelope, consistent with the critical role of ExoS/ChvI in growth and microbe-host interactions.