Cultivating the uncultured:Harnessing the“sandwich agar plate”approach to isolate heme-dependent bacteria from marine sediment

In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.

Quantitative comparisons of select cultured and uncultured microbial populations in the rumen of cattle fed different diets

Background： The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene firs） sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. Results： Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotello was about one log smaller than that of total bacteria. The populations of Fibrobocter succinogenes, Ruminococcus flovefociens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotello. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flovefociens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. Conclusions： Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.

The effect of long-term application of nitrogen-rich fertilizers on soil resistome:A study of conventional and organic cropping systems

Metagenomic studies of various soil environments have previously revealed the widespread distribution of antibiotic resistance genes(ARGs)around the globe.In this study,we applied shotgun metagenomics to investigate differences in microbial communities and resistomes in Chernozem soils that have been under long-term organic and conventional cropping practices.The organic cropping system was seeded with Triticum spelta without any fertilizer.The conventional cropping system was seeded with Tríticum durum Desf and used mineral fertilizer(NPK),that resulted in an increased amount of total and available carbon and nitrogen in soils.Across all samples,we identified a total of 21 ARG classes,among which the dominant were vancomycin,tetracycline and multidrug.Profiling of soil microbial communities revealed differences between the studied fields in the relative abundances of 14 and 53 genera in topsoil and subsoil,respectively.Correlation analysis showed significant correlations(positive and negative)among 18 genera and 6 ARGs,as well as between these ARGs and some chemical properties of soils.The analysis of metagenome-assembled genomes revealed that Nitrospirota,Thermoproteota,Actinobacteriota and Binatota phyla of archaea and bacteria serve as hosts for glycopeptide and fluoroquinolone/tetracycline ARGs.Collectively,the data obtained enrich knowledge about the consequences of human agricultural activities in terms of soil microbiome modification and highlight the role of nitrogen cycling taxa,including uncultivated genera,in the formation of soil resistome.

Strategies for culturing active/dormant marine microbes

Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service.However,many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture.Although many novel techniques have brought previously uncultured microbes into laboratory culture,there are still many most-wanted or key players that need to be cultured from marine environments.This review discusses possible reasons for‘unculturable microbes’and categorizes uncultured bacteria into three groups:dominant active bacteria,rare active bacteria,and dormant bacteria.This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria.Simulating the natural environment is an effective strategy for isolating dominant active bacteria,whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria.For dormant bacteria,resuscitation culture is an appropriate strategy.Furthermore,the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments.The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.