A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitro P. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.

To understand bacteria in diverse contexts, we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations.

Gaining insight into the behavior of bacteria at the single cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels both in liquid culture and in biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression was much stronger in anoxic environments compared to other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and demonstrate the utility of HCR in unveiling cellular activities at the microscale in heterogeneous populations.

Corals rely on a wide range of microorganisms for their functioning, including intracellular dinoflagellates (Symbiodiniaceae) and bacteria. Marine heatwaves trigger the loss of Symbiodiniaceae from coral tissues - coral bleaching - often leading to death. While coral-bacteria interactions are widely studied, Symbiodiniaceae-bacteria interactions have remained largely uninvestigated. Here, we provide a genomic analysis of 49 bacteria, spanning 16 genera, that closely associate with six cultured Symbiodiniaceae species. We analyzed bacterial functional potential by focusing on potentially beneficial functions for the Symbiodiniaceae host, including B vitamin synthesis and antioxidant abilities, which may be crucial for Symbiodiniaceae heat tolerance and in turn coral resistance to thermal bleaching. These analyses suggest a wide potential for B vitamin synthesis and the scavenging of reactive oxygen species (through the production of carotenoids or antioxidant enzymes), and possibly the transfer of organic carbon to host cells. Single nucleotide polymorphism analysis between bacteria isolated from wild-type and heat-evolved Symbiodiniaceae cultures revealed that exposure to long-term elevated temperature has resulted in mutations in genes known to be involved in host-symbiont interactions, such as secretion systems. Climate change may therefore modify how Symbiodiniaceae and bacteria interact. This study provides an overview of the possible roles of Symbiodiniaceae-associated bacteria in Symbiodiniaceae functioning and heat tolerance, reinforcing the need for further studies of such interactions to fully understand coral biology and climate resilience.