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Ziegler, C. A.

Publications and source records attributed to Ziegler, C. A..

3 recordsLinked to original sources

The shared and distinct roles of Prochlorococcus and co-occurring heterotrophic bacteria in regulating community dynamics

Prochlorococcus is the worlds most abundant photosynthetic organism with over 1027 cells distributed across much of Earths oceans, and is collectively responsible for almost 10% of marine carbon fixation. Naturally co-occurring heterotrophic bacteria at roughly 105-106 cells mL-1 in the oceans have been shown to increase Prochlorococcus fitness and productivity. Despite this massive scale, our understanding of these globally important interactions remains limited, with past research largely focused on single Prochlorococcus-heterotroph pairings involving only a few species. In this study, we extend this perspective by using synthetic communities containing multiple diverse heterotrophic strains isolated from Prochlorococcus enrichment cultures. Specifically, we isolated the four most abundant co-occurring heterotroph species and examined both individual Prochlorococcus-heterotroph interactions and interactions within a synthetic community comprising Prochlorococcus and all four heterotrophs. Using absolute quantification of RNA, DNA, and cell counts over the course of Prochlorococcus growth curves, we find that Prochlorococcus has a modest, species-independent transcriptional response to heterotrophs, whereas each heterotroph displays a markedly different transcriptional response to the community and fulfills distinct metabolic roles. Transcriptional analyses reveal several potential crossfeeding interactions and indicate that community dynamics are influenced not only by metabolic activity but also antagonistic mechanisms, defense responses, and coordinated group behaviors. By pairing synthetic community approaches with absolute abundance measurements, we can gain deeper insight into the forces that shape microbial community assembly in the oceans and their role in driving the global carbon cycle.

microbiology↗

Transformation of plasmid DNA into Prochlorococcus via electroporation

Prochlorococcus is the most numerically abundant photosynthetic organism in the oceans and plays a role in global carbon cycling. Despite its ecological significance and the availability of over a thousand assembled genomes, progress in understanding gene function has been limited by the lack of genetic tools. Here, we report a reproducible electroporation-based protocol to introduce replicative plasmids into two strains of Prochlorococcus representing different ecotypes: MIT9313 (low-light adapted) and MED4 (high-light adapted). Using plasmids carrying a spectinomycin resistance cassette, we achieved transformation in ~33% of MED4 and ~10% of MIT9313 attempts, with greatest success when electroporating cells in late exponential phase. Transformed cells stably retained plasmids and expressed resistance genes, demonstrating functional uptake and gene expression. We also delivered a modified 13 kb plasmid carrying a CRISPR-Cpf1 system into MED4. While no targeted edits were observed, cpf1 and specR were expressed, indicating successful delivery of large constructs and active transcription. These findings represent a key step toward genetic manipulation of Prochlorococcus, enabling future optimization of gene editing approaches and deeper functional analysis of its vast and largely uncharacterized pangenome.

molecular biology↗

E. coli Leucine-responsive regulatory protein bridges DNA in vivo and tunably dissociates in the presence of exogenous leucine

Feast-Famine Response Proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the E. coli leucine-responsive regulatory protein (Lrp). Lrp senses environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium towards the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrps regulatory activity, and that contrary to prior speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also find evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation.

microbiology↗