Lecture 19: Microbiology - Who does what, where, and why?

1. Rhizobia form symbiosis with legumes and fix atmospheric nitrogen to ammonia 2. Pathogens cause disease. e.g., salmonella 3. Phytoplankton drive marine carbon fixation and are important in DMSP synthesis 4. Actinomycetes e.g., Streptomyces, are important producers of antibiotics

• Take the bacteria that convert 90% of DMSP produced into DMS as an example a. Understand the basics - they eat DMS and take a methyl group as a source of carbon

• Culture-dependent work 2. Culture-independent work

• “Isolation and study of microbes that you can grow in a lab” a. take sample (seawater) plate it out on rich medium - yeast extract, provides everything needed • Oldest method, has lead to many microbiological findings • CON: Diverse mix of microbes, not specific How can you isolate the bacteria you want? 1. Use minimal media with DMS as sole C source 2. Some organisms, called “agaroses” can sink into the agar and release enzymes to use agar as C source

• Enrichment Culturing a. Inoculate bacteria to a liquid culture where DMS is only source of carbon b. move to new liquid DMS culture, keep repeating c. plate on DMS plates

• Enrichment Culturing a. Inoculate bacteria to a liquid culture where DMS is only source of carbon b. move to new liquid DMS culture, keep repeating c. plate on DMS plates

• Diagnostic genes (conserved between organisms) a. e.g., 16s RNA gene or 18s RNA gene

• model organisms that catabolise DMS (e.g., Methylophaga thioxydans DMS010) • Study physiology (DMS010 uses DMS and MeSH • Study genetics of DMS usage (e.g., gene involved, conservation in other bacteira, transcription/translation) • Essential if you desire progressing to biochemical understanding (Hard to do with culture-independent work) • NEED TO BE CAREFUL IT IS ONLY ONE BACTERIA

• Less than 1% of bacteria are cultivatable under lab conditions. Why? a. media missing unknown essential components b. microbes cannot grow axenically (on their own) c. microbes grow in liquid by not on plates d. Microbes gro too slowly e. Microbes may require growth in host cells (pathogens/symbionts) • Bacteria that grow in lab may not represent major players in the environment

• “Study of microbes in an environment/sample without their isolation on agar plates” Relatively newer methods, fully utilising high throughput sequencing and bioinformatics

1. Take sample of environment (ie. 1-20L of seawater or a g of soil) 2. Isolate microbes (e.g., filter 1-20L sample or spin down soil) 3. Isolate metagenomic DNA from microbial community microbes (e.g., carry out a gDNA prep) 4. Carry out 16s or 18s rRNA PCR on metagenomic DNA (community DNA) and observe single DNA species on agarose gel

• Denaturing Gradient Gel Electrophoresis (DGGE) - way of visualising distinct rRNA gene products (old fashioned) • High throughput sequencing - amplicon sequencing of the PCR product to show community diversity

• Allows direct visualisation of microbial diversity • Allows identification of major community members (low number) • Relatively cheap • Takes little time • Requires special 5’ primer with GC clamp • Separation of DNA frags. of same length with different BP sequences • Based on decreased electrophoretic mobility of a partially melted DNA molecule

+Works best for major bands +Gives idea of community complexity + Demonstrate if an enrichment has occurred in enrichment studies -Extremely tricky for outputs -If microbe is not abundant it may not give you any info

• Allow complete characterisation of all components of a microbial community depending on sequencing depth • E.g., can detect if a pathogen is in a sample at very low abundance • Now quite cheap (£50) and v. little time (month)

• Take sample • Prep gDNA • Carry out PCR on gDNA using 16S or 18S primers and purify PCR product • Amplicon sequence the product: a. Prepare a sequencing library (normally illumina) - a process that adds short DNA adaptors enabling sequencing b. Sequenced en masse (typically illumina MiSeq c. Given all the sequence reads ~30 million rRNA gene sequences • Bioinformatics analysis to classify different microbes in the community

Help identify what bugs within a sample of DGGE and amplicon sequencing catabolising DMS

• This is the process of applying high throughput sequencing on metagenomic DNA • Thus will give Taxonomic information on the community (based on all gene in data) • Also gives info on the metabolic potential of the community via indicating the abundance of diagnostic genes that drive processes. e.g., ddd genes for DMSP lysis • Has potential to identify the key microbes driving the process. E.g., the most abundant microbe that has your process gene

• Percent composition of an organism of a particular kind relative to the total number of organisms in the area • e.g., relative abundance of 80% means the Piscirickettsiaceae are predicted to comprise 80% of the bacteria in the sample • This can also be applied to genes, but in this case your gene of interest is normalised to genes that are known to be a single copy in genomes e.g., recA in bacteria