Transcriptome and Deletion Mutant Analyses Revealed that an RpoH Family Sigma Factor Is Essential for Photosystem Production in Roseateles depolymerans under Carbon Starvation.

Roseateles depolymerans is an obligately aerobic bacterium that produces a photosynthetic apparatus only under the scarcity of carbon substrates. We herein examined changes in the transcriptomes of R. depolymerans cells to clarify the expression of photosynthesis genes and their upstream regulatory factors under carbon starvation. Transcriptomes 0, 1, and 6| |h after the depletion of a carbon substrate indicated that transcripts showing the greatest variations (a 500-fold increase [6 h/0 h]) were light-harvesting proteins (PufA and PufB). Moreover, loci with more than 50-fold increases (6 h/0| |h) were fully related to the photosynthetic gene cluster. Among 13 sigma factor genes, the transcripts of a sigma 70 family sigma factor related to RpoH (SP70) increased along photosynthesis genes under starvation; therefore, a knockout experiment of SP70 was performed. ΔSP70 mutants were found to lack photosynthetic pigments (carotenoids and bacteriochlo-rophyll a) regardless of carbon starvation. We also examined the effects of heat stress on ΔSP70 mutants, and found that SP70 was also related to heat stress tolerance, similar to other RpoH sigma factors (while heat stress did not trigger photosystem production). The deficient accumulation of photosynthetic pigments and the heat stress tolerance of ΔSP70 mutants were both complemented by the introduction of an intact SP70 gene. Furthermore, the transcription of photosynthetic gene operons (puf, puh, and bch) was markedly reduced in the ΔSP70 mutant. The RpoH homologue SP70 was concluded to be a sigma factor that is essential for the transcription of photosynthetic gene operons in R. depolymerans.

Nondestructive microbial discrimination using single-cell Raman spectra and random forest machine learning algorithm.

Raman microspectroscopy is a powerful tool for obtaining biomolecular information from single microbial cells in a nondestructive manner. Here, we detail steps to discriminate prokaryotic species using single-cell Raman spectra acquisitions followed by data preprocessing and random forest model tuning. In addition, we describe the steps required to evaluate the model. This protocol requires minimal preprocessing of Raman spectral data, making it accessible to non-spectroscopists, yet allows intuitive visualization of feature importance. For complete details on the use and execution of this protocol, please refer to Kanno et al. (2021).

Resonance Raman analysis of intracellular vitamin B12 analogs in methanogenic archaea.

Methanogenic archaea (methanogens) are microorganisms that can synthesize methane. They are found in diverse environments ranging from paddy fields to animal digestive tracts to deep-sea hydrothermal vents. Investigating their distribution and physiological activity is crucial for the detailed analysis of the dynamics of greenhouse gas generation and the search for the environmental limits of life. In methanogens, cobamide cofactors (vitamin B12 analogs) play a key role in methane synthesis and carbon fixation, thus serving as a marker compound that metabolically characterizes them. Here, we report on resonance Raman detection of cobamides in methanogenic cells without destroying cells and provide structural insights into those cobamides. We succeeded in detecting cobamides in four representative methanogens Methanosarcina mazei, Methanosarcina barkeri, Methanopyrus kandleri, and Methanocaldococcus jannaschii. The former two are mesophilic, cytochrome-containing methanogens, whereas the latter two are hyperthermophilic, non-cytochrome-containing methanogens. The 532 nm-excited Raman spectra of single or multiple cells of the four species all showed resonance Raman bands of cobamides arising mainly from the corrin ring, with the most intense one at ∼1500 cm-1. We envision that resonance Raman microspectroscopy could be useful for in situ, nondestructive identification of methanogenic cells that produce high levels of cobamides.

Machine learning-assisted single-cell Raman fingerprinting for in situ and nondestructive classification of prokaryotes.

Accessing enormous uncultivated microorganisms (microbial dark matter) in various Earth environments requires accurate, nondestructive classification, and molecular understanding of the microorganisms in in situ and at the single-cell level. Here we demonstrate a combined approach of random forest (RF) machine learning and single-cell Raman microspectroscopy for accurate classification of phylogenetically diverse prokaryotes (three bacterial and three archaeal species from different phyla). Our RF classifier achieved a 98.8 ± 1.9% classification accuracy among the six species in pure populations and 98.4% for three species in an artificially mixed population. Feature importance scores against each wavenumber reveal that the presence of carotenoids and structure of membrane lipids play key roles in distinguishing the prokaryotic species. We also find unique Raman markers for an ammonia-oxidizing archaeon. Our approach with moderate data pretreatment and intuitive visualization of feature importance is easy to use for non-spectroscopists, and thus offers microbiologists a new single-cell tool for shedding light on microbial dark matter.

Monitoring of the toxic dinoflagellate Alexandrium catenella in Osaka Bay, Japan using a massively parallel sequencing (MPS)-based technique.

Since 2002, blooms of Alexandrium catenella sensu Fraga et al. (2015) and paralytic shellfish toxicity events have occurred almost yearly in Osaka Bay, Japan. To better understand the triggers for reoccurring A. catenella blooms in Osaka Bay, phytoplankton community was monitored during the spring seasons of 2012-2015. Monitoring was performed using massively parallel sequencing (MPS)-based technique on amplicon sequences of the 18S rRNA gene. Dense blooms of A. catenella occurred every year except in 2012, however, there was no significant correlation with the environmental parameters investigated. Plankton community diversity decreased before and middle of the A. catenella blooms, suggesting that the decline in diversity could be an indicator for the bloom occurrence. The yearly abundance pattern of A. catenella cells obtained by morphology-based counting coincided with the relative sequence abundances, which supports the effectiveness of MPS-based phytoplankton monitoring.

Sulfide-dependent Photoautotrophy in the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aggregans.

Chloroflexus aggregans is a thermophilic filamentous anoxygenic phototrophic bacterium frequently found in microbial mats in natural hot springs. C. aggregans often thrives with cyanobacteria that engage in photosynthesis to provide it with an organic substrate; however, it sometimes appears as the dominant phototroph in microbial mats without cyanobacteria. This suggests that C. aggregans has the ability to grow photoautotrophically. However, photoautotrophic growth has not been observed in any cultured strains of C. aggregans. We herein attempted to isolate a photoautotrophic strain from C. aggregansdominated microbial mats in Nakabusa hot spring in Japan. Using an inorganic medium, we succeeded in isolating a new strain that we designated "ACA-12". A phylogenetic analysis based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences revealed that strain ACA-12 was closely related to known C. aggregans strains. Strain ACA-12 showed sulfide consumption along with autotrophic growth under anaerobic light conditions. The deposited elemental sulfur particles observed by microscopy indicated that sulfide oxidation occurred, similar to that in photoautotrophic strains in the related species, C. aurantiacus. Moreover, we found that other strains of C. aggregans, including the type strain, also exhibited a slight photoautotrophic growing ability, whereas strain ACA-12 showed the fastest growth rate. This is the first demonstration of photoautotrophic growth with sulfide in C. aggregans. The present results strongly indicate that C. aggregans is associated with inorganic carbon incorporation using sulfide as an electron donor in hot spring microbial mats.

Toxic HAB species from the Sea of Okhotsk detected by a metagenetic approach, seasonality and environmental drivers.

During recent decades, the distribution of harmful algal bloom (HAB) species has expanded worldwide together with the increase of blooms and toxicity events. In this study, the presence of toxic HAB species in the Sea of Okhotsk was investigated based on metagenetic data collected during 6 years of weekly monitoring. Operational taxonomic units (OTUs) associated with the toxic HAB species were detected based on amplifying 18S V7-V9 and 28S D1 rRNA gene regions. In total, 43 unique OTUs associated with toxic HAB species were revealed, with 26 of those previously not reported from the Sea of Okhotsk. More OTUs belonging to dinoflagellates were detected by 18S, whereas a similar number of OTUs associated with dinoflagellates and diatoms were detected by targeting the 28S region. Species belonging to genera Alexandrium, Karenia and Karlodinium were mainly associated with OTUs under Dinophyceae, whereas Bacillariophyceae was represented by the species belonging to genus Pseudo-nitzschia. From the detected OTUs, 22 showed a clear seasonal pattern with the majority of those appearing during summer-autumn. For Alexandrium pacificum, Aureococcus anophagefferens, and Pseudo-nitzschia pungens, the seasonal pattern was detected based on both rRNA regions. Additionally, 14 OTUs were detected during all seasons and two OTUs appeared sporadically. OTUs associated with the toxic species had low relative read abundances, which together with other factors such as similar and variable morphology as well as usage of fixatives, may explain why those species have previously not been detected by light microscopy. Environmental parameters, especially water temperature, significantly (<0.05) influenced the variability in OTU relative abundances and displayed significant (<0.05) correlations with the unique OTUs. The results of this study demonstrate the usefulness of the metagenetic approach for phytoplankton monitoring, which is especially relevant for detecting toxic HAB species.

Genetic relatedness of a new Japanese isolates of Alexandrium ostenfeldii bloom population with global isolates.

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

Different Metabolomic Responses to Carbon Starvation between Light and Dark Conditions in the Purple Photosynthetic Bacterium, Rhodopseudomonas palustris.

Purple photosynthetic bacteria utilize light energy for growth. We previously demonstrated that light energy contributed to prolonging the survival of multiple purple bacteria under carbon-starved conditions. In order to clarify the effects of illumination on metabolic states under carbon-starved, non-growing conditions, we herein compared the metabolic profiles of starved cells in the light and dark using the purple bacterium, Rhodopseudomonas palustris. The metabolic profiles of starved cells in the light were markedly different from those in the dark. After starvation for 5 d in the light, cells showed increases in the amount of ATP and the NAD+/NADH ratio. Decreases in the amounts of most metabolites related to glycolysis and the TCA cycle in energy-rich starved cells suggest the active utilization of these metabolites for the modification of cellular components. Starvation in the dark induced the consumption of cellular compounds such as amino acids, indicating that the degradation of these cellular components produced ATP in order to maintain viability under energy-poor conditions. The present results suggest that intracellular energy levels alter survival strategies under carbon-starved conditions through metabolism.

Increase of Salt Tolerance in Carbon-Starved Cells of Rhodopseudomonas palustris Depending on Photosynthesis or Respiration.

Bacteria in natural environments are frequently exposed to nutrient starvation and survive against environmental stresses under non-growing conditions. In order to determine the energetic influence on survivability during starvation, changes in salt tolerance were investigated using the purple photosynthetic bacterium Rhodopseudomonas palustris after carbon starvation under photosynthetic conditions in comparison with anaerobic and aerobic dark conditions. Tolerance to a treatment with high concentration of salt (2.5 M NaCl for 1 h) was largely increased after starvation under anaerobically light and aerobically dark conditions. The starved cells under the conditions of photosynthesis or aerobic respiration contained high levels of cellular ATP, but starvation under the anaerobic dark conditions resulted in a decrease of cellular ATP contents. To observe the large increase of the salt tolerance, incubation of starved cells for more than 18 h under illumination was needed. These results suggest that the ATP-dependent rearrangement of cells induced salt tolerance.

Differences in Survivability under Starvation Conditions Among Four Species of Purple Nonsulfur Phototrophic Bacteria.

Volume 29, no. 3, Page 326-328, 2014Page 327, Legend for Fig. 1 IncorrectFig. 1. Changes in viability (black line) and ATP levels (gray line) during carbon-starvation conditions. CorrectFig. 1. Changes in viability (gray line) and ATP levels (black line) during carbon-starvation conditions.

Differences in survivability under starvation conditions among four species of purple nonsulfur phototrophic bacteria.

Survivability under carbon-starvation conditions was investigated in four species of purple phototrophic bacteria: Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodospirillum rubrum, and Rubrivivax gelatinosus. All these test organisms survived longer in the light than in the dark. ATP levels in the cultures were maintained in the light, which indicated that survivability was supported by photosynthesis. Survivability and tolerance against hypertonic stress in the dark was higher in Rhodopseudomonas palustris, which is widely distributed in natural environments including soils, than in the three other species.