In microbial mat communities of Yellowstone hot springs, ribosomal RNA (rRNA) sequence diversity patterns indicate the presence of closely related bacterial populations along environmental gradients of temperature and light. To identify the functional bases for adaptation, we sequenced the genomes of two cyanobacterial (Synechococcus OS-A and OS-B') isolates representing ecologically distinct populations that dominate at different temperatures and are major primary producers in the mat. There was a marked lack of conserved large-scale gene order between the two Synechococcus genomes, indicative of extensive genomic rearrangements. Comparative genomic analyses showed that the isolates shared a large fraction of their gene content at high identity, yet, differences in phosphate and nitrogen utilization pathways indicated that they have adapted differentially to nutrient fluxes, possibly by the acquisition of genes by lateral gene transfer or their loss in certain populations. Comparisons of the Synechococcus genomes to metagenomic sequences derived from mats where these Synechococcus stains were originally isolated, revealed new facets of microbial diversity. First, Synechococcus populations at the lower temperature regions of the mat showed greater sequence diversity than those at high temperatures, consistent with a greater number of ecologically distinct populations at the lower temperature. Second, we found evidence of a specialized population that is apparently very closely related to Synechococcus OS-B', but contains genes that function in the uptake of reduced ferrous iron. In situ expression studies demonstrated that these genes are differentially expressed over the diel cycle, with highest expression when the mats are anoxic and iron may be in the reduced state. Genomic information from these mat-specific isolates and metagenomic information can be coupled to detect naturally occurring populations that are associated with different functionalities, not always represented by isolates, but which may nevertheless be important for niche partitioning and the establishment of microbial community structure.

Background: Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics.

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium-Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.

Environmental cues can stimulate a variety of single-cell responses, as well as collective behaviors that emerge within a bacterial community. These responses require signal integration and transduction, which can occur on a variety of time scales and often involve feedback between processes, for example, between growth and motility. Here, we investigate the dynamics of responses of the phototactic, unicellular cyanobacterium Synechocystis sp. PCC6803 to complex light inputs that simulate the natural environments that cells typically encounter. We quantified single-cell motility characteristics in response to light of different wavelengths and intensities. We found that red and green light primarily affected motility bias rather than speed, while blue light inhibited motility altogether. When light signals were simultaneously presented from different directions, cells exhibited phototaxis along the vector sum of the light directions, indicating that cells can sense and combine multiple signals into an integrated motility response. Under a combination of antagonistic light signal regimes (phototaxis-promoting green light and phototaxis-inhibiting blue light), the ensuing bias was continuously tuned by competition between the wavelengths, and the community response was dependent on both bias and cell growth. The phototactic dynamics upon a rapid light shift revealed a wavelength dependence on the time scales of photoreceptor activation/deactivation. Thus, Synechocystis cells achieve exquisite integration of light inputs at the cellular scale through continuous tuning of motility, and the pattern of collective behavior depends on single-cell motility and population growth.

To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.

Despite extensive DNA sequencing data derived from natural microbial communities, it remains a major challenge to identify the key evolutionary and ecological forces that shape microbial populations. We have focused on the extensive microdiversity of the cyanobacterium Synechococcus sp., which is a dominant member of the dense phototrophic biofilms in the hot springs of Yellowstone National Park. From deep amplicon sequencing of many loci and statistical analyses of these data, we showed previously that the population has undergone an unexpectedly high degree of homologous recombination, unlinking synonymous SNP-pair correlations even on intragenic length scales. Here, we analyze the genic amino acid diversity, which provides new evidence of selection and insights into the evolutionary history of the population. Surprisingly, some features of the data, including the spectrum of distances between genic-alleles, appear consistent with primarily asexual neutral drift. Yet the non-synonymous site frequency spectrum has too large an excess of low-frequency polymorphisms to result from negative selection on deleterious mutations given the distribution of coalescent times that we infer. And our previous analyses showed that the population is not asexual. Taken together, these apparently contradictory data suggest that selection, epistasis, and hitchhiking all play essential roles in generating and stabilizing the diversity. We discuss these as well as potential roles of ecological niches at genomic and genic levels. From quantitative properties of the diversity and comparative genomic data, we infer aspects of the history and inter-spring dispersal of the meta-population since it was established in the Yellowstone Caldera. Our investigations illustrate the need for combining multiple types of sequencing data and quantitative statistical analyses to develop an understanding of microdiversity in natural microbial populations.

To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high, GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc10 and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.

Polyphosphate (polyP), a polymer of orthophosphate (PO43-) of varying lengths, has been identified in all kingdoms of life. It can serve as a source of chemical bond energy (phosphoanhydride bond) that may have been used by biological systems prior to the evolution of ATP. Intracellular polyP is mainly stored as granules in specific vacuoles called acidocalcisomes, and its synthesis and accumulation appear to impact a myriad of cellular functions. It serves as a reservoir for inorganic PO(4)(3-)and an energy source for fueling cellular metabolism, participates in maintaining adenylate and metal cation homeostasis, functions as a scaffold for sequestering cations, exhibits chaperone function, covalently binds to proteins to modify their activity, and enables normal acclimation of cells to stress conditions. PolyP also appears to have a role in symbiotic and parasitic associations, and in higher eukaryotes, low polyP levels seem to impact cancerous proliferation, apoptosis, procoagulant and proinflammatory responses and cause defects in TOR signaling. In this review, we discuss the metabolism, storage, and function of polyP in photosynthetic microbes, which mostly includes research on green algae and cyanobacteria. We focus on factors that impact polyP synthesis, specific enzymes required for its synthesis and degradation, sequestration of polyP in acidocalcisomes, its role in cellular energetics, acclimation processes, and metal homeostasis, and then transition to its potential applications for bioremediation and medical purposes.