Lecture 21:Rare and Uncultured Microbes

Microbiology 480Prof. Kristen DeAngelis

Spring 2016

Objectives

• You should be able to recognize these phyla, their important characteristics, and example species– Deinococci, Chlamydia & Planctomycetes

• What is reductive evolution? metabolic engineering?

• How well do cultivated species represent diversity? Why?

• What is the Eocyte hypothesis, and how does this tree differ from the three domain tree?

Deinococci, Chlamydia & Planctomycetes

Phylum Deinococcus-Thermus

Deinococcus and relatives• Diversity of the order Deinococcus is low, with a

small collection (~18) very closely related species• Metabolism

– Aerobic heterotrophs– Mostly mesophiles– Extreme resistance to DNA-damaging stress: UV

radiation, desiccation, oxidizing agents, and mutagens• Habitat

– Found in soil, sediment, rainwater, sewage, dust– Rare members of these communities– Most isolates from irradiated samples (food, clean

rooms, and nuclear reactor cooling pools)

Deinococcus radiodurans

Thermus and relatives

• More diversity than in the order Deinococcus with four genera and many isolates

• Metabolism– Thermophilic heterotrophs– Oligotrophs, prefer low nutrient concentrations– Obligate aerobes or facultative anaerobes

• Habitat– Neutral to slightly alkaline hot springs– 55oC – 80oC– Some halophiles, some deep sea hydrothermal vents

Thermus aquaticus

• Isolated from many alkaline hot springs in Yellowstone NP

• Is often pink or white especially when grown in light conditions due to pigments

• Its DNA polymerase is highly heat resistant and error-correcting– Taq polymerase is commonly

used in PCR

Phylum Chlamydiae

Phylum Chlamydiae• Low phylogenetic and phenotypic diversity

– Few cultured representatives– Uncultured diversity seems to be much greater

• Metabolism– Greatly reduced genomes– Remain capable of

• information processing (transcription, translation, replication)• cell envelope• central metabolism

• Habitat: Obligate intracellular parasites transmitted via small, metabolically inert particles

Developmental cycle of Chlamydia

Chlamydia trachomatisPhylum Chlamydiae

• Human pathogen that causes the most common sexually transmitted disease (STD) in the U.S.

• Most infections are asymptomatic, but untreated infections can cause sterility

• Repeated ocular infection in children can cause blindness

C. trachomatis elementary bodies attached to human sperm. From Courtney S. Hossenzadeh in Microbiology Today.

Phylum Planctomycetes

Fig. 13.12 P. bekefii

Fig. 13.13 P. bekefii

Phylum Planctomycetes

• Diversity is unclear, because they are rarely cultivated despite their unique morphology

• Metabolism of almost all are aerobic, heterotrophic, mesophilic oligotrophs

• Habitats are mostly aquatic and especially eutrophic environments, though sequences are detected in a wide range of environments including wastewater and soils

• Example: Blastopirellula marina

Blastopirellula marinaPhylum Planctomycetes

• Common freshwater species• Has the simplest

compartmentalization of the planctomycetes

• All planctomycetes have some internal membrane-defined compartmentalization– Central pirellulosome contains the

riboplasm and nucleoid (genome)– Riboplasm contains ribosomes and DNA– Paryphoplasm contains RNA but not

ribosomes

Reductive evolution• Over evolutionary time, parasites rely more on the

host for the things it needs and may simplify its genome

• This reduction allows the organism to devote more resources to reproduction

• Examples of reductive evolution in bacteria include – Chlamydia: phylum of intracellular parasites– Rikettsias: genus of class Alphaproteobacteria, include

mitochondria– Mycoplasmas: genus of phylum Firmicutes capable of

gliding motility

Reductive metabolic engineering

• first minimal synthetic bacterial cell, JCVI-syn3.0• Mycoplasma mycoides was tested for non-essential genes, and these

were systematically removed– new minimal synthetic cell contains 531,560 base pairs and just 473 genes,

with a doubling time of 90 mins– Starting cell M. mycoides (phylum Firmicutes) had 1.2 Mbp genome and had

a doubling time of 7 hours– M. mycoides is an intracellular parasite so it’s genome was already reduced

• 32% of the genes essential for life in this cell are of unknown function

Bacterial phyla with few to no

cultivated representatives

Classic view of the Tree of Life

Woese, Candler & Wheelis. 1990. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. PNAS

How can we expand known diversity?

1. Cultivation and enrichment

2. Molecular techniques

Constructing a phylogenetic treeAssume you have chosen species to analyze1. Decide which gene to use (SSU rRNA gene)2. Determine the gene sequences3. Use sequence alignment to identify homologous

residues4. Measure sequence similarity and make a

distance matrix 5. Perform phylogenetic analysis, which usually

means constructing a tree

SSU ribosomal RNA gene+ Short, only 1500 base pairs+ Information-dense because it is a non-coding,

structural RNA+ Essential for life so probably not horizontally

transferred- Multiple copies per genome- Cannot resolve close relationships

16S ribosomal RNA primers

Kuczynski et al 2012, Nature Reviews Genetics 13, 47-58

Sequence alignment

Bacterial phyla with few to no

cultivated representatives

Rare but functionally dominant

• 13 “main” bacterial phyla in black• Common assumption that numerical dominance

= functional importance• Rare but functionally dominant• Examples

– Nitrospira: aquatic N cycle as nitrite oxidizers– Acidobacteria: soil acidophilic heterotrophs– Verrucomicrobia: soil heterotrophs, possibly dominant

and underestimated because of primer bias

Molecular approaches aka ‘Omics

Kuczynski et al 2012, Nature Reviews Genetics 13, 47-58

Molecular approaches aka ‘Omics• Genomics – sequencing whole genomes• Metagenomics – sequencing mixed communities• Transcriptomics – sequencing RNA from genomes• Metatranscriptomics – sequencing RNA from mixed communities

Assembling whole genomes from metagenomic data using binning

• Puts shotgun sequences into “bins” based on shared characteristics– GC content– Coverage– K-mer frequency– taxonomy

http://dx.doi.org/10.3389/fmicb.2015.01451

New Tree of Life

Slides thanks to Jonathan Eisen! @phylogenomics

New View of the Domain Bacteria

• The phyla we study make up a small portion of the total estimated phylogenetic diversity

• The total functional diversity is still unknown.

Hug et al. 2016. Nature Microbiology.

Bacteroids & Spirochetes

Hug et al. 2016. Nature Microbiology.

Green phototrophic bacteria

Hug et al. 2016. Nature Microbiology.

Thermophilic bacteria

Hug et al. 2016. Nature Microbiology.

Phylum Proteobacteria

Hug et al. 2016. Nature Microbiology.

Gram-positive bacteria

Hug et al. 2016. Nature Microbiology.

Deinococci, Chlamydiae & Planctomycetes

Hug et al. 2016. Nature Microbiology.

Phyla never grown in the labHug et al. 2016. Nature Microbiology.

Slides thanks to Jonathan Eisen! @phylogenomics

Eocyte hypothesis

• This two-domain Eocyte tree and the three-domain tree are competing hypotheses for the origin of Eukarya.

• The Eocyte hypothesis is a biological classification that indicates eukaryotes evolved from the prokaryotic Crenarchaeota (formerly known as eocytes), a phylum within the archaea.

• This hypothesis was based on the discovery that the shapes of ribosomes in the Crenarchaeota and eukaryotes are more similar to each other than to either bacteria or the second major kingdom of archaea, the Euryarchaeota.

Archaea & Eukaryotes

• Most archaeal phyla have never been grown in the lab!

Hug et al. 2016. Nature Microbiology.

Objectives

• You should be able to recognize these phyla, their important characteristics, and example species– Deinococci, Chlamydia & Planctomycetes

• What is reductive evolution? metabolic engineering?

• How well do cultivated species represent diversity? Why?

• What is the Eocyte hypothesis, and how does this tree differ from the three domain tree?