4.1: Proteobacteria - Biology

Learning Objectives

  • Describe the unique features of each class within the phylum Proteobacteria: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria
  • Give an example of a bacterium in each class of Proteobacteria

In 1987, the American microbiologist Carl Woese (1928–2012) suggested that a large and diverse group of bacteria that he called “purple bacteria and their relatives” should be defined as a separate phylum within the domain Bacteria based on the similarity of the nucleotide sequences in their genome.1 This phylum of gram-negative bacteria subsequently received the name Proteobacteria. It includes many bacteria that are part of the normal human microbiota as well as many pathogens. The Proteobacteria are further divided into five classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria.


The first class of Proteobacteria is the Alphaproteobacteria. The unifying characteristic of this class is that they are oligotrophs, organisms capable of living in low-nutrient environments such as deep oceanic sediments, glacial ice, or deep undersurface soil.

Among the Alphaproteobacteria are two taxa, chlamydias and rickettsias, that are obligate intracellular pathogens, meaning that part of their life cycle must occur inside other cells called host cells. When not growing inside a host cell, Chlamydia and Rickettsia are metabolically inactive outside of the host cell. They cannot synthesize their own adenosine triphosphate (ATP), and, therefore, rely on cells for their energy needs.

Rickettsia spp. include a number of serious human pathogens. For example, R. rickettsii causes Rocky Mountain spotted fever, a life-threatening form of meningoencephalitis (inflammation of the membranes that wrap the brain). R. rickettsii infects ticks and can be transmitted to humans via a bite from an infected tick (Figure (PageIndex{1})).

Another species of Rickettsia, R. prowazekii, is spread by lice. It causes epidemic typhus, a severe infectious disease common during warfare and mass migrations of people. prowazekii infects human endothelium cells, causing inflammation of the inner lining of blood vessels, high fever, abdominal pain, and sometimes delirium. A relative, R. typhi, causes a less severe disease known as murine or endemic typhus, which is still observed in the southwestern United States during warm seasons.

Chlamydia is another taxon of the Alphaproteobacteria. Members of this genus are extremely resistant to the cellular defenses, giving them the ability to spread from host to host rapidly via elementary bodies. The metabolically and reproductively inactive elementary bodies are the endospore-like form of intracellular bacteria that enter an epithelial cell, where they become active. Figure (PageIndex{2}) illustrates the life cycle of Chlamydia.

C. trachomatis is a human pathogen that causes trachoma, a disease of the eyes, often leading to blindness. C. trachomatis also causes the sexually transmitted disease lymphogranuloma venereum (LGV). This disease is often mildly symptomatic, manifesting as regional lymph node swelling, or it may be asymptomatic, but it is extremely contagious and is common on college campuses.

Table (PageIndex{1}) summarizes the characteristics of important genera of Alphaproteobacteria.Table (PageIndex{1}): Class Alphaproteobacteria
GenusMicroscopic MorphologyUnique Characteristics
AgrobacteriumGram-negative bacillusPlant pathogen; one species, A. tumefaciens, causes tumors in plants
BartonellaGram-negative, pleomorphic, flagellated coccobacillusFacultative intracellular bacteria, transmitted by lice and fleas, cause trench fever and cat scratch disease in humans
BrucellaGram-negative, small, flagellated coccobacillusFacultative intracellular bacteria, transmitted by contaminated milk from infected cows, cause brucellosis in cattle and humans
CaulobacterGram-negative bacillusUsed in studies on cellular adaptation and differentiation because of its peculiar life cycle (during cell division, forms “swarm” cells and “stalked” cells)
ChlamydiaGram-negative, coccoid or ovoid bacteriumObligatory intracellular bacteria; some cause chlamydia, trachoma, and pneumonia
CoxiellaSmall, gram-negative bacillusObligatory intracellular bacteria; cause Q fever; potential for use as biological weapon
EhrlichiaVery small, gram-negative, coccoid or ovoid bacteriaObligatory intracellular bacteria; can be transported from cell to cell; transmitted by ticks; cause ehrlichiosis (destruction of white blood cells and inflammation) in humans and dogs
HyphomicrobiumGram-negative bacilli; grows from a stalkSimilar to Caulobacter
MethylocystisGram-negative, coccoid or short bacilliNitrogen-fixing aerobic bacteria
RhizobiumGram-negative, rectangular bacilli with rounded ends forming clustersNitrogen-fixing bacteria that live in soil and form symbiotic relationship with roots of legumes (e.g., clover, alfalfa, and beans)
RickettsiaGram-negative, highly pleomorphic bacteria (may be cocci, rods, or threads)Obligate intracellular bacteria; transmitted by ticks; may cause Rocky Mountain spotted fever and typhus

Exercise (PageIndex{1})

What characteristic do all Alphaproteobacteria share?


Unlike Alphaproteobacteria, which survive on a minimal amount of nutrients, the class Betaproteobacteria are eutrophs (or copiotrophs), meaning that they require a copious amount of organic nutrients. Betaproteobacteria often grow between aerobic and anaerobic areas (e.g., in mammalian intestines). Some genera include species that are human pathogens, able to cause severe, sometimes life-threatening disease. The genus Neisseria, for example, includes the bacteria N. gonorrhoeae, the causative agent of the STI gonorrhea, and N. meningitides, the causative agent of bacterial meningitis.

Neisseria are cocci that live on mucosal surfaces of the human body. They are fastidious, or difficult to culture, and they require high levels of moisture, nutrient supplements, and carbon dioxide. Also, Neisseria are microaerophilic, meaning that they require low levels of oxygen. For optimal growth and for the purposes of identification, Neisseria spp. are grown on chocolate agar (i.e., agar supplemented by partially hemolyzed red blood cells). Their characteristic pattern of growth in culture is diplococcal: pairs of cells resembling coffee beans (Figure (PageIndex{3})).

The pathogen responsible for pertussis (whooping cough) is also a member of Betaproteobacteria. The bacterium Bordetella pertussis, from the order Burkholderiales, produces several toxins that paralyze the movement of cilia in the human respiratory tract and directly damage cells of the respiratory tract, causing a severe cough.

Table (PageIndex{2}) summarizes the characteristics of important genera of Betaproteobacteria.Table (PageIndex{2}): Class Betaproteobacteria
Example GenusMicroscopic MorphologyUnique Characteristics
BordetellaA small, gram-negative coccobacillusAerobic, very fastidious; B. pertussis causes pertussis (whooping cough)
BurkholderiaGram-negative bacillusAerobic, aquatic, cause diseases in horses and humans (especially patients with cystic fibrosis); agents of nosocomial infections
LeptothrixGram-negative, sheathed, filamentous bacillusAquatic; oxidize iron and manganese; can live in wastewater treatment plants and clog pipes
NeisseriaGram-negative, coffee bean-shaped coccus forming pairsRequire moisture and high concentration of carbon dioxide; oxidase positive, grow on chocolate agar; pathogenic species cause gonorrhea and meningitis
ThiobacillusGram-negative bacillusThermophilic, acidophilic, strictly aerobic bacteria; oxidize iron and sulfur

Exercise (PageIndex{2})

What characteristic do all Betaproteobacteria share?


When Marsha finally went to the doctor’s office, the physician listened to her breathing through a stethoscope. He heard some crepitation (a crackling sound) in her lungs, so he ordered a chest radiograph and asked the nurse to collect a sputum sample for microbiological evaluation and cytology. The radiologic evaluation found cavities, opacities, and a particular pattern of distribution of abnormal material (Figure (PageIndex{4})).

Exercise (PageIndex{3})

What are some possible diseases that could be responsible for Marsha’s radiograph results?


The most diverse class of gram-negative bacteria is Gammaproteobacteria, and it includes a number of human pathogens. For example, a large and diverse family, Pseudomonaceae, includes the genus Pseudomonas. Within this genus is the species P. aeruginosa, a pathogen responsible for diverse infections in various regions of the body. P. aeruginosa is a strictly aerobic, nonfermenting, highly motile bacterium. It often infects wounds and burns, can be the cause of chronic urinary tract infections, and can be an important cause of respiratory infections in patients with cystic fibrosis or patients on mechanical ventilators. Infections by P. aeruginosa are often difficult to treat because the bacterium is resistant to many antibiotics and has a remarkable ability to form biofilms. Other representatives of Pseudomonas include the fluorescent (glowing) bacterium P. fluorescens and the soil bacteria P. putida, which is known for its ability to degrade xenobiotics (substances not naturally produced or found in living organisms).

The Pasteurellaceae also includes several clinically relevant genera and species. This family includes several bacteria that are human and/or animal pathogens. For example, Pasteurella haemolytica causes severe pneumonia in sheep and goats. multocida is a species that can be transmitted from animals to humans through bites, causing infections of the skin and deeper tissues. The genus Haemophilus contains two human pathogens, H. influenzae and H. ducreyi. Despite its name, H. influenzae does not cause influenza (which is a viral disease). H. influenzae can cause both upper and lower respiratory tract infections, including sinusitis, bronchitis, ear infections, and pneumonia. Before the development of effective vaccination, strains of H. influenzae were a leading cause of more invasive diseases, like meningitis in children. ducreyi causes the STI known as chancroid.

The order Vibrionales includes the human pathogen Vibrio cholerae. This comma-shaped aquatic bacterium thrives in highly alkaline environments like shallow lagoons and sea ports. A toxin produced by V. cholerae causes hypersecretion of electrolytes and water in the large intestine, leading to profuse watery diarrhea and dehydration. V. parahaemolyticus is also a cause of gastrointestinal disease in humans, whereas V. vulnificus causes serious and potentially life-threatening cellulitis (infection of the skin and deeper tissues) and blood-borne infections. Another representative of Vibrionales, Aliivibrio fischeri, engages in a symbiotic relationship with squid. The squid provides nutrients for the bacteria to grow and the bacteria produce bioluminescence that protects the squid from predators (Figure (PageIndex{5})).

The genus Legionella also belongs to the Gammaproteobacteria. L. pneumophila, the pathogen responsible for Legionnaires disease, is an aquatic bacterium that tends to inhabit pools of warm water, such as those found in the tanks of air conditioning units in large buildings (Figure (PageIndex{6})). Because the bacteria can spread in aerosols, outbreaks of Legionnaires disease often affect residents of a building in which the water has become contaminated with Legionella. In fact, these bacteria derive their name from the first known outbreak of Legionnaires disease, which occurred in a hotel hosting an American Legion veterans’ association convention in Philadelphia in 1976.

Enterobacteriaceae is a large family of enteric (intestinal) bacteria belonging to the Gammaproteobacteria. They are facultative anaerobes and are able to ferment carbohydrates. Within this family, microbiologists recognize two distinct categories. The first category is called the coliforms, after its prototypical bacterium species, Escherichia coli. Coliforms are able to ferment lactose completely (i.e., with the production of acid and gas). The second category, noncoliforms, either cannot ferment lactose or can only ferment it incompletely (producing either acid or gas, but not both). The noncoliforms include some notable human pathogens, such as Salmonella spp., Shigella spp., and Yersinia pestis.

E. coli has been perhaps the most studied bacterium since it was first described in 1886 by Theodor Escherich (1857–1911). Many strains of E. coli are in mutualistic relationships with humans. However, some strains produce a potentially deadly toxin called Shiga toxin, which perforates cellular membranes in the large intestine, causing bloody diarrhea and peritonitis (inflammation of the inner linings of the abdominal cavity). Other E. coli strains may cause traveler’s diarrhea, a less severe but very widespread disease.

The genus Salmonella, which belongs to the noncoliform group of Enterobacteriaceae, is interesting in that there is still no consensus about how many species it includes. Scientists have reclassified many of the groups they once thought to be species as serotypes (also called serovars), which are strains or variations of the same species of bacteria. Their classification is based on patterns of reactivity by animal antisera against molecules on the surface of the bacterial cells. A number of serotypes of Salmonella can cause salmonellosis, characterized by inflammation of the small and the large intestine, accompanied by fever, vomiting, and diarrhea. The species S. enterobacterica (serovar typhi) causes typhoid fever, with symptoms including fever, abdominal pain, and skin rashes (Figure (PageIndex{7})).

Table (PageIndex{3}) summarizes the characteristics of important genera of Gammaproteobacteria.Table (PageIndex{3}): Class Gammaproteobacteria
Example GenusMicroscopic MorphologyUnique Characteristics
BeggiatoaGram-negative bacteria; disc-shaped or cylindricalAquatic, live in water with high content of hydrogen disulfide; can cause problems for sewage treatment
EnterobacterGram-negative bacillusFacultative anaerobe; cause urinary and respiratory tract infections in hospitalized patients; implicated in the pathogenesis of obesity
ErwiniaGram-negative bacillusPlant pathogen causing leaf spots and discoloration; may digest cellulose; prefer relatively low temperatures (25–30 °C)
EscherichiaGram-negative bacillusFacultative anaerobe; inhabit the gastrointestinal tract of warm-blooded animals; some strains are mutualists, producing vitamin K; others, like serotype E. coli O157:H7, are pathogens; E. coli has been a model organism for many studies in genetics and molecular biology
HemophilusGram-negative bacillusPleomorphic, may appear as coccobacillus, aerobe, or facultative anaerobe; grow on blood agar; pathogenic species can cause respiratory infections, chancroid, and other diseases
KlebsiellaGram-negative bacillus; appears rounder and thicker than other members of EnterobacteriaceaeFacultative anaerobe, encapsulated, nonmotile; pathogenic species may cause pneumonia, especially in people with alcoholism
LegionellaGram-negative bacillusFastidious, grow on charcoal-buffered yeast extract; L. pneumophila causes Legionnaires disease
MethylomonasGram-negative bacillusUse methane as source of carbon and energy
ProteusGram-negative bacillus (pleomorphic)Common inhabitants of the human gastrointestinal tract; motile; produce urease; opportunistic pathogens; may cause urinary tract infections and sepsis
PseudomonasGram-negative bacillusAerobic; versatile; produce yellow and blue pigments, making them appear green in culture; opportunistic, antibiotic-resistant pathogens may cause wound infections, hospital-acquired infections, and secondary infections in patients with cystic fibrosis
SerratiaGram-negative bacillusMotile; may produce red pigment; opportunistic pathogens responsible for a large number of hospital-acquired infections
ShigellaGram-negative bacillusNonmotile; dangerously pathogenic; produce Shiga toxin, which can destroy cells of the gastrointestinal tract; can cause dysentery
VibrioGram-negative, comma- or curved rod-shaped bacteriaInhabit seawater; flagellated, motile; may produce toxin that causes hypersecretion of water and electrolytes in the gastrointestinal tract; some species may cause serious wound infections
YersiniaGram-negative bacillusCarried by rodents; human pathogens; Y. pestis causes bubonic plague and pneumonic plague; Y. enterocolitica can be a pathogen causing diarrhea in humans

Exercise (PageIndex{4})

List two families of Gammaproteobacteria.


The Deltaproteobacteria is a small class of gram-negative Proteobacteria that includes sulfate-reducing bacteria(SRBs), so named because they use sulfate as the final electron acceptor in the electron transport chain. Few SRBs are pathogenic. However, the SRB Desulfovibrio orale is associated with periodontal disease (disease of the gums).

Deltaproteobacteria also includes the genus Bdellovibrio, species of which are parasites of other gram-negative bacteria. Bdellovibrio invades the cells of the host bacterium, positioning itself in the periplasm, the space between the plasma membrane and the cell wall, feeding on the host’s proteins and polysaccharides. The infection is lethal for the host cells.

Another type of Deltaproteobacteria, myxobacteria, lives in the soil, scavenging inorganic compounds. Motile and highly social, they interact with other bacteria within and outside their own group. They can form multicellular, macroscopic “fruiting bodies” (Figure (PageIndex{8})), structures that are still being studied by biologists and bacterial ecologists.2These bacteria can also form metabolically inactive myxospores.

Table (PageIndex{4}) summarizes the characteristics of several important genera of Deltaproteobacteria.Table (PageIndex{4}): Class Deltaproteobacteria
GenusMicroscopic MorphologyUnique characteristics
BdellovibrioGram-negative, comma-shaped rodObligate aerobes; motile; parasitic (infecting other bacteria)
Desulfovibrio(formerly Desufuromonas)Gram-negative, comma-shaped rodReduce sulfur; can be used for removal of toxic and radioactive waste
MyxobacteriumGram-negative, coccoid bacteria forming colonies (swarms)Live in soil; can move by gliding; used as a model organism for studies of intercellular communication (signaling)

Exercise (PageIndex{5})

What type of Deltaproteobacteria forms fruiting bodies?


The smallest class of Proteobacteria is Epsilonproteobacteria, which are gram-negative microaerophilic bacteria (meaning they only require small amounts of oxygen in their environment). Two clinically relevant genera of Epsilonproteobacteria are Campylobacter and Helicobacter, both of which include human pathogens. Campylobacter can cause food poisoning that manifests as severe enteritis (inflammation in the small intestine). This condition, caused by the species C. jejuni, is rather common in developed countries, usually because of eating contaminated poultry products. Chickens often harbor C. jejuni in their gastrointestinal tract and feces, and their meat can become contaminated during processing.

Within the genus Helicobacter, the helical, flagellated bacterium H. pylori has been identified as a beneficial member of the stomach microbiota, but it is also the most common cause of chronic gastritis and ulcers of the stomach and duodenum (Figure (PageIndex{9})). Studies have also shown that H. pylori is linked to stomach cancer.3 H. pylori is somewhat unusual in its ability to survive in the highly acidic environment of the stomach. It produces urease and other enzymes that modify its environment to make it less acidic.

Table (PageIndex{5}) summarizes the characteristics of the most clinically relevant genera of Epsilonproteobacteria.Table (PageIndex{5}): Class Epsilonproteobacteria
Example GenusMicroscopic MorphologyUnique Characteristics
CampylobacterGram-negative, spiral-shaped rodAerobic (microaerophilic); often infects chickens; may infect humans via undercooked meat, causing severe enteritis
HelicobacterGram-negative, spiral-shaped rodAerobic (microaerophilic) bacterium; can damage the inner lining of the stomach, causing chronic gastritis, peptic ulcers, and stomach cancer

Exercise (PageIndex{1})

Name two Epsilonproteobacteria that cause gastrointestinal disorders.


  • Proteobacteria is a phylum of gram-negative bacteria discovered by Carl Woese in the 1980s based on nucleotide sequence homology.
  • Proteobacteria are further classified into the classes alpha-, beta-, gamma-, delta- and epsilonproteobacteria, each class having separate orders, families, genera, and species.
  • Alphaproteobacteria are oligotrophs. The taxa chlamydias and rickettsias are obligate intracellular pathogens, feeding on cells of host organisms; they are metabolically inactive outside of the host cell. Some Alphaproteobacteria can convert atmospheric nitrogen to nitrites, making nitrogen usable by other forms of life.
  • Betaproteobacteria are eutrophs. They include human pathogens of the genus Neisseria and the species Bordetella pertussis.
  • Gammaproteobacteria are the largest and the most diverse group of Proteobacteria. Many are human pathogens that are aerobes or facultative anaerobes. Some Gammaproteobacteria are enteric bacteria that may be coliform or noncoliform. Escherichia coli, a member of Gammaproteobacteria, is perhaps the most studied bacterium.
  • Deltaproteobacteria make up a small group able to reduce sulfate or elemental sulfur. Some are scavengers and form myxospores, with multicellular fruiting bodies.
  • Epsilonproteobacteria make up the smallest group of Proteobacteria. The genera Campylobacter and Helicobacter are human pathogens.
  1. 1 C.R. Woese. “Bacterial Evolution.” Microbiological Review 51 no. 2 (1987):221–271.
  2. 2 H. Reichenbach. “Myxobacteria, Producers of Novel Bioactive Substances.” Journal of Industrial Microbiology & Biotechnology 27 no. 3 (2001):149–156.
  3. 3 S. Suerbaum, P. Michetti. “Helicobacter pylori infection.” New England Journal of Medicine 347 no. 15 (2002):1175–1186.


  • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at

Illumina sequencing-based analysis of free-living bacterial community dynamics during an Akashiwo sanguine bloom in Xiamen sea, China

Although phytoplankton are the major source of marine dissolved organic matter (DOM), their blooms are a global problem that can greatly affect marine ecological systems, especially free-living bacteria, which are the primary DOM degraders. In this study, we analyzed free-living bacterial communities from Xiamen sea during an Akashiwo sanguine bloom using Illumina MiSeq sequencing of 16S rRNA gene amplicons. The bloom was probably stimulated by low salinity and ended after abatement of eutrophication pollution. A total of 658,446 sequence reads and 11,807 OTUs were obtained in both bloom and control samples with Alpha-proteobacteria and Gamma-proteobacteria being the predominant classes detected. The bloom decreased bacterial diversity, increased species evenness, and significantly changed the bacterial community structure. Bacterial communities within the bloom were more homogeneous than those within the control area. The bacteria stimulated by this bloom included the SAR86 and SAR116 clades and the AEGEAN-169 marine group, but a few were suppressed. In addition, many bacteria known to be associated with phytoplankton were detected only in the bloom samples. This study revealed the great influence of an A. sanguinea bloom on free-living bacterial communities, and provided new insights into the relationship between bacteria and A. sanguinea in marine ecosystems.


Figure 1. Location of sampling sites.

Figure 1. Location of sampling sites.

This map was created based on PhotoShop (Version CS5)…

Figure 2. Rarefaction curves of bloom and…

Figure 2. Rarefaction curves of bloom and control samples.

Figure 3. Composition of the top 10…

Figure 3. Composition of the top 10 taxa at the (A) class, (B) order, (C)…

Figure 4. Rarefaction curves (A) and species…

Figure 4. Rarefaction curves (A) and species richness (B) estimates for bloom and control samples…

Figure 5. Bacterial community structure at the…

Figure 5. Bacterial community structure at the genus level.

Only the top 10 genera are…

Figure 6. Detrended correspondence analysis (DCA) of…

Figure 6. Detrended correspondence analysis (DCA) of the bacterial community.

Figure 7. Free-living bacterial Simpson evenness of…

Figure 7. Free-living bacterial Simpson evenness of the bloom and control groups.

Figure 8. Unclassified sequence ratios of bloom…

Figure 8. Unclassified sequence ratios of bloom and control areas at the genus level.

Figure 9. Heat map illustrating the-fold change…

Figure 9. Heat map illustrating the-fold change of genera identified in comparisons between the bloom…

Figure 10. Relative abundance of bacterial genera…

Figure 10. Relative abundance of bacterial genera in the bloom and control areas.


All "Proteobacteria" are Gram-negative (though some may stain Gram-positive or Gram-variable in practice), with an outer membrane mainly composed of lipopolysaccharides. Many move about using flagella, but some are nonmotile or rely on bacterial gliding. The latter include the myxobacteria, an order of bacteria that can aggregate to form multicellular fruiting bodies. Also, a wide variety in the types of metabolism exists. Most members are facultatively or obligately anaerobic, chemolithoautotrophic, and heterotrophic, but numerous exceptions occur. A variety of genera, which are not closely related to each other, convert energy from light through photosynthesis.

"Proteobacteria" are associated with the imbalance of microbiota of the lower reproductive tract of women. These species are associated with inflammation. ⎚]

Riboregulation in plant-associated α-proteobacteria

The symbiotic α-rhizobia Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium etli and the related plant pathogen Agrobacterium tumefaciens are important model organisms for studying plant-microbe interactions. These metabolically versatile soil bacteria are characterized by complex lifestyles and large genomes. Here we summarize the recent knowledge on their small non-coding RNAs (sRNAs) including conservation, function, and interaction of the sRNAs with the RNA chaperone Hfq. In each of these organisms, an inventory of hundreds of cis- and trans-encoded sRNAs with regulatory potential was uncovered by high-throughput approaches and used for the construction of 39 sRNA family models. Genome-wide analyses of hfq mutants and co-immunoprecipitation with tagged Hfq revealed a major impact of the RNA chaperone on the physiology of plant-associated α-proteobacteria including symbiosis and virulence. Highly conserved members of the SmelC411 family are the AbcR sRNAs, which predominantly regulate ABC transport systems. AbcR1 of A. tumefaciens controls the uptake of the plant-generated signaling molecule GABA and is a central regulator of nutrient uptake systems. It has similar functions in S. meliloti and the human pathogen Brucella abortus. As RNA degradation is an important process in RNA-based gene regulation, a short overview on ribonucleases in plant-associated α-proteobacteria concludes this review.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.


This work was supported by the following grants from the Deutsche Forschungsgemeinschaft (DFG) in the framework of the priority program SPP 1258 “Sensory and Regulatory RNAs in Prokaryotes”: BE 2121/3–1, BE 2121/4–1, BE 2121/5–2, FN 240/8–1, GI 178/4–1, GI 178/4–2, Ev42/4–1, and Ev42/4–2.

4.1 Prokaryote Habitats, Relationships, and Microbiomes

Marsha, a 20-year-old university student, recently returned to the United States from a trip to Nigeria, where she had interned as a medical assistant for an organization working to improve access to laboratory services for tuberculosis testing. When she returned, Marsha began to feel fatigue, which she initially attributed to jet lag. However, the fatigue persisted, and Marsha soon began to experience other bothersome symptoms, such as occasional coughing, night sweats, loss of appetite, and a low-grade fever of 37.4 °C (99.3 °F).

Marsha expected her symptoms would subside in a few days, but instead, they gradually became more severe. About two weeks after returning home, she coughed up some sputum and noticed that it contained blood and small whitish clumps resembling cottage cheese. Her fever spiked to 38.2 °C (100.8 °F), and she began feeling sharp pains in her chest when breathing deeply. Concerned that she seemed to be getting worse, Marsha scheduled an appointment with her physician.

  • Could Marsha’s symptoms be related to her overseas travel, even several weeks after returning home?

Jump to the next Clinical Focus box.

All living organisms are classified into three domains of life: Archaea , Bacteria , and Eukarya . In this chapter, we will focus on the domains Archaea and Bacteria. Archaea and bacteria are unicellular prokaryotic organisms. Unlike eukaryotes, they have no nuclei or any other membrane-bound organelles.

Prokaryote Habitats and Functions

Prokaryotes are ubiquitous. They can be found everywhere on our planet, even in hot springs, in the Antarctic ice shield, and under extreme pressure two miles under water. One bacterium, Paracoccus denitrificans, has even been shown to survive when scientists removed it from its native environment (soil) and used a centrifuge to subject it to forces of gravity as strong as those found on the surface of Jupiter.

Prokaryotes also are abundant on and within the human body. According to a report by National Institutes of Health, prokaryotes , especially bacteria, outnumber human cells 10:1. 1 More recent studies suggest the ratio could be closer to 1:1, but even that ratio means that there are a great number of bacteria within the human body. 2 Bacteria thrive in the human mouth, nasal cavity, throat, ears, gastrointestinal tract, and vagina. Large colonies of bacteria can be found on healthy human skin, especially in moist areas (armpits, navel, and areas behind ears). However, even drier areas of the skin are not free from bacteria.

The existence of prokaryotes is very important for the stability and thriving of ecosystems. For example, they are a necessary part of soil formation and stabilization processes through the breakdown of organic matter and development of biofilms. One gram of soil contains up to 10 billion microorganisms (most of them prokaryotic) belonging to about 1,000 species. Many species of bacteria use substances released from plant roots, such as acids and carbohydrates, as nutrients. The bacteria metabolize these plant substances and release the products of bacterial metabolism back to the soil, forming humus and thus increasing the soil’s fertility. In salty lakes such as the Dead Sea (Figure 4.2), salt-loving halobacteria decompose dead brine shrimp and nourish young brine shrimp and flies with the products of bacterial metabolism.

In addition to living in the ground and the water, prokaryotic microorganisms are abundant in the air, even high in the atmosphere. There may be up to 2,000 different kinds of bacteria in the air, similar to their diversity in the soil.

Prokaryotes can be found everywhere on earth because they are extremely resilient and adaptable. They are often metabolically flexible, which means that they might easily switch from one energy source to another, depending on the availability of the sources, or from one metabolic pathway to another. For example, certain prokaryotic cyanobacteria can switch from a conventional type of lipid metabolism, which includes production of fatty aldehydes, to a different type of lipid metabolism that generates biofuel, such as fatty acids and wax esters. Groundwater bacteria store complex high-energy carbohydrates when grown in pure groundwater, but they metabolize these molecules when the groundwater is enriched with phosphates. Some bacteria get their energy by reducing sulfates into sulfides, but can switch to a different metabolic pathway when necessary, producing acids and free hydrogen ions.

Prokaryotes perform functions vital to life on earth by capturing (or “fixing”) and recycling elements like carbon and nitrogen. Organisms such as animals require organic carbon to grow, but, unlike prokaryotes, they are unable to use inorganic carbon sources like carbon dioxide. Thus, animals rely on prokaryotes to convert carbon dioxide into organic carbon products that they can use. This process of converting carbon dioxide to organic carbon products is called carbon fixation .

Plants and animals also rely heavily on prokaryotes for nitrogen fixation , the conversion of atmospheric nitrogen into ammonia, a compound that some plants can use to form many different biomolecules necessary to their survival. Bacteria in the genus Rhizobium , for example, are nitrogen-fixing bacteria they live in the roots of legume plants such as clover, alfalfa, and peas (Figure 4.3). Ammonia produced by Rhizobium helps these plants to survive by enabling them to make building blocks of nucleic acids. In turn, these plants may be eaten by animals—sustaining their growth and survival—or they may die, in which case the products of nitrogen fixation will enrich the soil and be used by other plants.

Another positive function of prokaryotes is in cleaning up the environment. Recently, some researchers focused on the diversity and functions of prokaryotes in manmade environments. They found that some bacteria play a unique role in degrading toxic chemicals that pollute water and soil. 3

Despite all of the positive and helpful roles prokaryotes play, some are human pathogens that may cause illness or infection when they enter the body. In addition, some bacteria can contaminate food, causing spoilage or foodborne illness, which makes them subjects of concern in food preparation and safety. Less than 1% of prokaryotes (all of them bacteria) are thought to be human pathogens, but collectively these species are responsible for a large number of the diseases that afflict humans.

Besides pathogens, which have a direct impact on human health, prokaryotes also affect humans in many indirect ways. For example, prokaryotes are now thought to be key players in the processes of climate change . In recent years, as temperatures in the earth’s polar regions have risen, soil that was formerly frozen year-round (permafrost) has begun to thaw. Carbon trapped in the permafrost is gradually released and metabolized by prokaryotes. This produces massive amounts of carbon dioxide and methane, greenhouse gases that escape into the atmosphere and contribute to the greenhouse effect.

Check Your Understanding

  • In what types of environments can prokaryotes be found?
  • Name some ways that plants and animals rely on prokaryotes.

Symbiotic Relationships

As we have learned, prokaryotic microorganisms can associate with plants and animals. Often, this association results in unique relationships between organisms. For example, bacteria living on the roots or leaves of a plant get nutrients from the plant and, in return, produce substances that protect the plant from pathogens. On the other hand, some bacteria are plant pathogens that use mechanisms of infection similar to bacterial pathogens of animals and humans.

Prokaryotes live in a community , or a group of interacting populations of organisms. A population is a group of individual organisms belonging to the same biological species and limited to a certain geographic area. Populations can have cooperative interactions , which benefit the populations, or competitive interactions , in which one population competes with another for resources. The study of these interactions between microbial populations and their environment is called microbial ecology .

Any interaction between different species that are associated with each other within a community is called symbiosis . Such interactions fall along a continuum between opposition and cooperation. Interactions in a symbiotic relationship may be beneficial or harmful, or have no effect on one or both of the species involved. Table 4.1 summarizes the main types of symbiotic interactions among prokaryotes.

Types of Symbiotic Relationships
Type Population A Population B
Mutualism Benefitted Benefitted
Amensalism Harmed Unaffected
Commensalism Benefitted Unaffected
Neutralism Unaffected Unaffected
Parasitism Benefitted Harmed

When two species benefit from each other, the symbiosis is called mutualism (or syntropy, or crossfeeding). For example, humans have a mutualistic relationship with the bacterium Bacteroides thetaiotaomicron, which lives in the intestinal tract. Bacteroides thetaiotaomicron digests complex polysaccharide plant materials that human digestive enzymes cannot break down, converting them into monosaccharides that can be absorbed by human cells. Humans also have a mutualistic relationship with certain strains of Escherichia coli , another bacterium found in the gut. E. coli relies on intestinal contents for nutrients, and humans derive certain vitamins from E. coli, particularly vitamin K, which is required for the formation of blood clotting factors. (This is only true for some strains of E. coli, however. Other strains are pathogenic and do not have a mutualistic relationship with humans.)

A type of symbiosis in which one population harms another but remains unaffected itself is called amensalism . In the case of bacteria, some amensalist species produce bactericidal substances that kill other species of bacteria. The microbiota of the skin is composed of a variety of bacterial species, including Staphylococcus epidermidis and Propionibacterium acnes. Although both species have the potential to cause infectious diseases when protective barriers are breached, they both produce a variety of antibacterial bacteriocins and bacteriocin-like compounds. S. epidermidis and P. acnes are unaffected by the bacteriocins and bacteriocin-like compounds they produce, but these compounds can target and kill other potential pathogens.

In another type of symbiosis, called commensalism , one organism benefits while the other is unaffected. This occurs when the bacterium Staphylococcus epidermidis uses the dead cells of the human skin as nutrients. Billions of these bacteria live on our skin, but in most cases (especially when our immune system is healthy), we do not react to them in any way. S. epidermidis provides an excellent example of how the classifications of symbiotic relationships are not always distinct. One could also consider the symbiotic relationship of S. epidermidis with humans as mutualism. Humans provide a food source of dead skin cells to the bacterium, and in turn the production of bacteriocin can provide an defense against potential pathogens.

If neither of the symbiotic organisms is affected in any way, we call this type of symbiosis neutralism . An example of neutralism is the coexistence of metabolically active (vegetating) bacteria and endospores (dormant, metabolically passive bacteria). For example, the bacterium Bacillus anthracis typically forms endospores in soil when conditions are unfavorable. If the soil is warmed and enriched with nutrients, some B. anthracis endospores germinate and remain in symbiosis with other species of endospores that have not germinated.

A type of symbiosis in which one organism benefits while harming the other is called parasitism . The relationship between humans and many pathogenic prokaryotes can be characterized as parasitic because these organisms invade the body, producing toxic substances or infectious diseases that cause harm. Diseases such as tetanus, diphtheria, pertussis, tuberculosis, and leprosy all arise from interactions between bacteria and humans.

Scientists have coined the term microbiome to refer to all prokaryotic and eukaryotic microorganisms and their genetic material that are associated with a certain organism or environment. Within the human microbiome , there are resident microbiota and transient microbiota . The resident microbiota consists of microorganisms that constantly live in or on our bodies. The term transient microbiota refers to microorganisms that are only temporarily found in the human body, and these may include pathogenic microorganisms. Hygiene and diet can alter both the resident and transient microbiota.

The resident microbiota is amazingly diverse, not only in terms of the variety of species but also in terms of the preference of different microorganisms for different areas of the human body. For example, in the human mouth, there are thousands of commensal or mutualistic species of bacteria. Some of these bacteria prefer to inhabit the surface of the tongue, whereas others prefer the internal surface of the cheeks, and yet others prefer the front or back teeth or gums. The inner surface of the cheek has the least diverse microbiota because of its exposure to oxygen. By contrast, the crypts of the tongue and the spaces between teeth are two sites with limited oxygen exposure, so these sites have more diverse microbiota, including bacteria living in the absence of oxygen (e.g., Bacteroides , Fusobacterium ). Differences in the oral microbiota between randomly chosen human individuals are also significant. Studies have shown, for example, that the prevalence of such bacteria as Streptococcus , Haemophilus , Neisseria , and others was dramatically different when compared between individuals. 4

There are also significant differences between the microbiota of different sites of the same human body. The inner surface of the cheek has a predominance of Streptococcus, whereas in the throat, the palatine tonsil, and saliva, there are two to three times fewer Streptococcus, and several times more Fusobacterium. In the plaque removed from gums, the predominant bacteria belong to the genus Fusobacterium. However, in the intestine, both Streptococcus and Fusobacterium disappear, and the genus Bacteroides becomes predominant.

Not only can the microbiota vary from one body site to another, the microbiome can also change over time within the same individual. Humans acquire their first inoculations of normal flora during natural birth and shortly after birth. Before birth, there is a rapid increase in the population of Lactobacillus spp. in the vagina, and this population serves as the first colonization of microbiota during natural birth. After birth, additional microbes are acquired from health-care providers, parents, other relatives, and individuals who come in contact with the baby. This process establishes a microbiome that will continue to evolve over the course of the individual’s life as new microbes colonize and are eliminated from the body. For example, it is estimated that within a 9-hour period, the microbiota of the small intestine can change so that half of the microbial inhabitants will be different. 5 The importance of the initial Lactobacillus colonization during vaginal child birth is highlighted by studies demonstrating a higher incidence of diseases in individuals born by cesarean section , compared to those born vaginally. Studies have shown that babies born vaginally are predominantly colonized by vaginal lactobacillus, whereas babies born by cesarean section are more frequently colonized by microbes of the normal skin microbiota, including common hospital-acquired pathogens.

Throughout the body, resident microbiotas are important for human health because they occupy niches that might be otherwise taken by pathogenic microorganisms. For instance, Lactobacillus spp. are the dominant bacterial species of the normal vaginal microbiota for most women. lactobacillus produce lactic acid, contributing to the acidity of the vagina and inhibiting the growth of pathogenic yeasts. However, when the population of the resident microbiota is decreased for some reason (e.g., because of taking antibiotics), the pH of the vagina increases, making it a more favorable environment for the growth of yeasts such as Candida albicans . Antibiotic therapy can also disrupt the microbiota of the intestinal tract and respiratory tract, increasing the risk for secondary infections and/or promoting the long-term carriage and shedding of pathogens.

Check Your Understanding

  • Explain the difference between cooperative and competitive interactions in microbial communities.
  • List the types of symbiosis and explain how each population is affected.

Taxonomy and Systematics

Assigning prokaryotes to a certain species is challenging. They do not reproduce sexually, so it is not possible to classify them according to the presence or absence of interbreeding. Also, they do not have many morphological features. Traditionally, the classification of prokaryotes was based on their shape, staining patterns, and biochemical or physiological differences. More recently, as technology has improved, the nucleotide sequences in genes have become an important criterion of microbial classification.

In 1923, American microbiologist David Hendricks Bergey (1860–1937) published A Manual in Determinative Bacteriology. With this manual, he attempted to summarize the information about the kinds of bacteria known at that time, using Latin binomial classification. Bergey also included the morphological, physiological, and biochemical properties of these organisms. His manual has been updated multiple times to include newer bacteria and their properties. It is a great aid in bacterial taxonomy and methods of characterization of bacteria. A more recent sister publication, the five-volume Bergey’s Manual of Systematic Bacteriology, expands on Bergey’s original manual. It includes a large number of additional species, along with up-to-date descriptions of the taxonomy and biological properties of all named prokaryotic taxa. This publication incorporates the approved names of bacteria as determined by the List of Prokaryotic Names with Standing in Nomenclature (LPSN).

Link to Learning

The 7th edition (published in 1957) of Bergey’s Manual of Determinative Bacteriology is now available. More recent, updated editions are available in print. You can also access a searchable database of microbial reference strains, published by the American Type Culture Collection (ATCC).

Classification by Staining Patterns

According to their staining patterns , which depend on the properties of their cell walls, bacteria have traditionally been classified into gram-positive, gram-negative, and “atypical,” meaning neither gram-positive nor gram-negative. As explained in Staining Microscopic Specimens, gram-positive bacteria possess a thick peptidoglycan cell wall that retains the primary stain (crystal violet) during the decolorizing step they remain purple after the gram-stain procedure because the crystal violet dominates the light red/pink color of the secondary counterstain, safranin. In contrast, gram-negative bacteria possess a thin peptidoglycan cell wall that does not prevent the crystal violet from washing away during the decolorizing step therefore, they appear light red/pink after staining with the safranin. Bacteria that cannot be stained by the standard Gram stain procedure are called atypical bacteria . Included in the atypical category are species of Mycoplasma and Chlamydia . Rickettsia are also considered atypical because they are too small to be evaluated by the Gram stain.

More recently, scientists have begun to further classify gram-negative and gram-positive bacteria. They have added a special group of deeply branching bacteria based on a combination of physiological, biochemical, and genetic features. They also now further classify gram-negative bacteria into Proteobacteria , Cytophaga-Flavobacterium-Bacteroides (CFB) , and spirochetes .

The deeply branching bacteria are thought to be a very early evolutionary form of bacteria (see Deeply Branching Bacteria). They live in hot, acidic, ultraviolet-light-exposed, and anaerobic (deprived of oxygen) conditions. Proteobacteria is a phylum of very diverse groups of gram-negative bacteria it includes some important human pathogens (e.g., E. coli and Bordetella pertussis ). The CFB group of bacteria includes components of the normal human gut microbiota, like Bacteroides . The spirochetes are spiral-shaped bacteria and include the pathogen Treponema pallidum , which causes syphilis. We will characterize these groups of bacteria in more detail later in the chapter.

Based on their prevalence of guanine and cytosine nucleotides, gram-positive bacteria are also classified into low G+C and high G+C gram-positive bacteria . The low G+C gram-positive bacteria have less than 50% of guanine and cytosine nucleotides in their DNA. They include human pathogens, such as those that cause anthrax ( Bacillus anthracis ), tetanus ( Clostridium tetani ), and listeriosis ( Listeria monocytogenes ). High G+C gram-positive bacteria, which have more than 50% guanine and cytosine nucleotides in their DNA, include the bacteria that cause diphtheria ( Corynebacterium diphtheriae ), tuberculosis ( Mycobacterium tuberculosis ), and other diseases.

The classifications of prokaryotes are constantly changing as new species are being discovered. We will describe them in more detail, along with the diseases they cause, in later sections and chapters.

Check Your Understanding

Micro Connections

Human Microbiome Project

The Human Microbiome Project was launched by the National Institutes of Health (NIH) in 2008. One main goal of the project is to create a large repository of the gene sequences of important microbes found in humans, helping biologists and clinicians understand the dynamics of the human microbiome and the relationship between the human microbiota and diseases. A network of labs working together has been compiling the data from swabs of several areas of the skin, gut, and mouth from hundreds of individuals.

One of the challenges in understanding the human microbiome has been the difficulty of culturing many of the microbes that inhabit the human body. It has been estimated that we are only able to culture 1% of the bacteria in nature and that we are unable to grow the remaining 99%. To address this challenge, researchers have used metagenomic analysis , which studies genetic material harvested directly from microbial communities, as opposed to that of individual species grown in a culture. This allows researchers to study the genetic material of all microbes in the microbiome, rather than just those that can be cultured. 6

One important achievement of the Human Microbiome Project is establishing the first reference database on microorganisms living in and on the human body. Many of the microbes in the microbiome are beneficial, but some are not. It was found, somewhat unexpectedly, that all of us have some serious microbial pathogens in our microbiota. For example, the conjunctiva of the human eye contains 24 genera of bacteria and numerous pathogenic species. 7 A healthy human mouth contains a number of species of the genus Streptococcus , including pathogenic species S. pyogenes and S. pneumoniae. 8 This raises the question of why certain prokaryotic organisms exist commensally in certain individuals but act as deadly pathogens in others. Also unexpected was the number of organisms that had never been cultured. For example, in one metagenomic study of the human gut microbiota, 174 new species of bacteria were identified. 9

Another goal for the near future is to characterize the human microbiota in patients with different diseases and to find out whether there are any relationships between the contents of an individual’s microbiota and risk for or susceptibility to specific diseases. Analyzing the microbiome in a person with a specific disease may reveal new ways to fight diseases.


    Medical Press. “Mouth Bacteria Can Change Their Diet, Supercomputers Reveal.” August 12, 2014. Accessed February 24, 2015. A. Abbott. “Scientists Bust Myth That Our Bodies Have More Bacteria Than Human Cells: Decades-Old Assumption about Microbiota Revisited.” Nature. Accessed June 3, 2016. A.M. Kravetz “Unique Bacteria Fights Man-Made Chemical Waste.” 2012. Accessed March 9, 2015. E.M. Bik et al. “Bacterial Diversity in the Oral Cavity of 10 Healthy Individuals.” The ISME Journal 4 no. 8 (2010):962–974. C.C. Booijink et al. “High Temporal and Intra-Individual Variation Detected in the Human Ileal Microbiota.” Environmental Microbiology 12 no. 12 (2010):3213–3227. National Institutes of Health. “Human Microbiome Project. Overview.” Accessed June 7, 2016. Q. Dong et al. “Diversity of Bacteria at Healthy Human Conjunctiva.” Investigative Ophthalmology & Visual Science 52 no. 8 (2011):5408–5413. F.E. Dewhirst et al. “The Human Oral Microbiome.” Journal of Bacteriology 192 no. 19 (2010):5002–5017. J.C. Lagier et al. “Microbial Culturomics: Paradigm Shift in the Human Gut Microbiome Study.” Clinical Microbiology and Infection 18 no. 12 (2012):1185–1193.

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    Bacterial small RNAs have been identified as components of many regulatory networks. Twenty sRNAs were experimentally identified in Rhodobacter spheroids, and the abundant ones were shown to be affected by singlet oxygen ( 1 O2) exposure. [6] 1 O2 which generates photooxidative stress, is made by bacteriochlorophyll upon exposure to oxygen and light. One of the 1 O2 induced sRNAs SorY ( 1 O2 resistance RNA Y) was shown to be induced under several stress conditions and conferred resistance against 1 O2 by affecting a metabolite transporter. [7] SorX is the second 1 O2 induced sRNA that counteracts oxidative stress by targeting mRNA for a transporter. It also has an impact on resistance against organic hydroperoxides. [8] A cluster of four homologous sRNAs called CcsR for conserved CCUCCUCCC motif stress-induced RNA has been shown to play a role in photo-oxidative stress resistance as well. [9] PcrZ (photosynthesis control RNA Z) identified in R. sphaeroides, is a trans-acting sRNA which counteracts the redox-dependent induction of photosynthesis genes, mediated by protein regulators. [10]

    R. sphaeroides encodes several terminal oxidases which allow electron transfer to oxygen and other electron acceptors (e.g. DMSO or TMAO). [11] Therefore, this microorganism can respire under oxic, micro-oxic and anoxic conditions under both light and dark conditions. Moreover, it is capable to accept a variety of carbon substrates, including C1 to C4 molecules, sugars and fatty acids. [12] Several pathways for glucose catabolism are present in its genome, such as the Embden–Meyerhof–Parnas pathway (EMP), the Entner–Doudoroff pathway (ED) and the Pentose phosphate pathway (PP). [13] The ED pathway is the predominant glycolytic pathway in this microorganism, [14] whereas the EMP pathway contributing only to a smaller extent. [15] Variation in nutrient availability has important effects on the physiology of this bacterium. For example, decrease in oxygen tensions activates the synthesis of photosynthetic machinery (including photosystems, antenna complexes and pigments). Moreover, depletion of nitrogen in the medium triggers intracellular accumulation of polyhydroxybutyrate, a reserve polymer. [16]

    A genome-scale metabolic model exists for this microorganism, [17] which can be used for predicting the effect of gene manipulations on its metabolic fluxes. For facilitating genome editing in this species, a CRISPR/Cas9 genome editing tool was developed [18] and expanded. [19] Moreover, partitioning of intracellular fluxes has been studied in detail, also with the help of 13 C-glucose isotopomers. [15] [20] Altogether, these tools can be employed for improving R. sphaeroides as cell factory for industrial biotechnology. [3]


    Production of NADH by Chemolithotrophic Bacteria

    With many chemolithotrophs, the potential of the half-reaction that drives metabolism (in the case of Nitrosomonas, the dehydrogenation of hydroxylamine) is apparently not low enough to reduce NAD + directly. Thus, chemoautotrophs must drive electrons in the reverse direction to reduce NAD + to NADH + H + in a process called reverse electron transport. The final reaction in this process is catalyzed by the NADH dehydrogenase. In chemoorganotrophs, the NADH dehydrogenase oxidizes NADH + H + to NAD + , reduces quinine, and pumps protons to the outside of the cell. However, in the case of Nitrosomonas ( Figure 2 ) and other chemolithotrophs, the reaction runs in the reverse direction to form NADH. This process is not needed in bacteria, such as the hydrogen oxidizers, where the growth-supporting potential of the reductive half reaction ( H 2 → 2 H + + 2 e − ) has a low enough value that NAD + is reduced directly.

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    In: Microbial Ecology , Vol. 63, No. 3, 01.04.2012, p. 522-531.

    Research output : Contribution to journal › Article › peer-review

    T1 - Growth Stimulation of Iodide-Oxidizing α-Proteobacteria in Iodide-Rich Environments

    N2 - α-Proteobacteria that can oxidize iodide (I -) to molecular iodine (I 2) have only been isolated from iodide-rich natural and artificial environments, i. e., natural gas brine waters and seawaters supplemented with iodide, respectively. To understand the growth characteristics of such iodide-oxidizing bacteria (IOB) under iodide-rich environments, microcosms comprising natural seawater and 1 mM iodide were prepared, and the succession of microbial communities was monitored by culture-independent techniques. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis showed that bacteria closely related with known IOB were predominant in the microcosms after several weeks of incubation. Quantitative PCR analysis targeting specific 16S rRNA gene regions of IOB showed that the relative abundance of IOB in the microcosms was 6-76% of the total bacterial population, whereas that in natural seawater was less than 1%. When 10 3 cells mL -1 of IOB were inoculated into natural seawater supplemented with 0. 1-1 mM iodide, significant growth (cell densities, 10 5-10 6 cells mL -1) and I 2 production (6-32 μM) were observed. Interestingly, similar growth stimulation occurred when 12-44 μM of I 2 was added to seawater, instead of iodide. IOB were found to be more I 2 tolerant than the other heterotrophic bacteria in seawater. These results suggest that I 2 plays a key role in the growth stimulation of IOB in seawater. IOB could potentially attack other bacteria with I 2 to occupy their ecological niche in iodide-rich environments.

    AB - α-Proteobacteria that can oxidize iodide (I -) to molecular iodine (I 2) have only been isolated from iodide-rich natural and artificial environments, i. e., natural gas brine waters and seawaters supplemented with iodide, respectively. To understand the growth characteristics of such iodide-oxidizing bacteria (IOB) under iodide-rich environments, microcosms comprising natural seawater and 1 mM iodide were prepared, and the succession of microbial communities was monitored by culture-independent techniques. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis showed that bacteria closely related with known IOB were predominant in the microcosms after several weeks of incubation. Quantitative PCR analysis targeting specific 16S rRNA gene regions of IOB showed that the relative abundance of IOB in the microcosms was 6-76% of the total bacterial population, whereas that in natural seawater was less than 1%. When 10 3 cells mL -1 of IOB were inoculated into natural seawater supplemented with 0. 1-1 mM iodide, significant growth (cell densities, 10 5-10 6 cells mL -1) and I 2 production (6-32 μM) were observed. Interestingly, similar growth stimulation occurred when 12-44 μM of I 2 was added to seawater, instead of iodide. IOB were found to be more I 2 tolerant than the other heterotrophic bacteria in seawater. These results suggest that I 2 plays a key role in the growth stimulation of IOB in seawater. IOB could potentially attack other bacteria with I 2 to occupy their ecological niche in iodide-rich environments.


    In the first half of the Results section, we address the first question of whether McrBC-mediated cell death through cleavage of methylated chromosomes takes place upon entry/induction of an epigenetic methyltransferase gene and causes this gene's establishment/activation to be aborted.

    McrBC-mediated inhibition of establishment of a DNA methyltransferase gene

    We first asked about the biological consequences of McrBC, that is, whether or not establishment of a transferred methyltransferase gene is aborted through the action of McrBC. As the methyltransferase, we chose PvuII methyltransferase (M.PvuII) of the PvuII RM system. It recognizes CAGCTG and generates CAG m4 CTG [37, 41], a target sequence of McrBC [33].

    Several reports have indicated that phages or plasmids carrying a DNA methyltransferase gene could not be propagated in an mcrBC + strain of E. coli [42]. Whether the block to propagation is due to repeated methylation of the introduced DNA and subsequent cleavage [42] or due to host genome methylation and cleavage, as we have hypothesized in this work, has not been addressed.

    We introduced a plasmid carrying the PvuII methyltransferase (M. PvuII, CAG m4 CTG) gene but lacking PvuII recognition sites (pEF43 in Table 1) in a quantitative transformation assay (Figure 2a). The transformation efficiency decreased by four orders of magnitude in an mcrBC-dependent manner (Figure 2b). The decrease did not occur in the case of genes for three other cytosine methyltransferases, M.EcoRII (C m5 CWGG), M.SsoII (C m5 CNGG), and M.BamHI (GGAT m4 CC), consistent with the sequence specificity of McrBC [33]. We found that a plasmid carrying a PvuII methyltransferase gene and two PvuII recognition sites was also inhibited in its establishment by the same order of magnitude (date not shown). Our results indicate that methylated sites on the transferred DNA were not required for the McrBC-dependent inhibition of its establishment and propagation. These results demonstrate that McrBC can abort establishment of the transferred element with the methyltransferase gene and, furthermore, suggest that this is through McrBC-mediated cleavage of methylated chromosomal DNA, as opposed to that on the transferred DNA.

    McrBC-mediated blocking of establishment of an epigenetic genome methylation system. (a) Quantitative transformation. Varying amounts of pUC19 (2 pg, 20 pg, 200 pg, 2 ng, 20 ng, and 200 ng) were used to transform E. coli DH5α by electroporation. Experiments were conducted in triplicate. (b) Transformation of plasmids carrying the PvuII methyltransferase gene. Plasmids (100 ng) carrying one of several modification methyltransferase genes were used to transform E. coli ER1562 (mcrB1) and ER1563 (mcrBC + ). The relative transformation efficiency was calculated as the ratio of the transformation efficiency of the test plasmid to that of the empty vector. M.PvuII (ColE1) indicates pEF43, while M.PvuII (pPvu1) indicates pEF65 (Table 1). The empty vector for the latter is pEF67, while that for the former is pEF33. The vector for the remaining plasmids is pBR322. The measurements from two separate experiments conducted in duplicate are shown. All (20/20) of the rare transformants of mcrBC + by pEF43 examined were found to have lost McrBC activity.

    The PvuII RM gene complex was found on pPvu1, a low-copy plasmid from Proteus vulgaris [37] that can also replicate in E. coli [43]. Proteus vulgaris and E. coli both belong to the Enterobacteriaceae family and also share an ecological niche, the intestine of humans and related animals. Therefore, these experiments are intended to reproduce events that are likely to take place in the natural environment, although they involved the use of multicopy (ColE1-derived) plasmids. Transformation of a pPvu1 derivative plasmid carrying M.PvuII and a drug-resistance gene as a selective marker and lacking PvuII sites (pEF65 in Table 1) was blocked by McrBC as strongly as the above multi-copy plasmid (Figure 2b). This suggests that the strong inhibition is biologically relevant.

    McrBC-mediated chromosome cleavage after phage-mediated transfer of the DNA methyltransferase gene

    The above inhibition of establishment of the methyltransferase gene is likely caused by lethal cleavage of chromosomes that become methylated. Next, we asked whether McrBC indeed cleaves host chromosomes in order to abort the propagation of a transfered epigenetic genome methylation gene. In order to examine this issue, we introduced the M.PvuII gene into E. coli by a λ phage vector.

    We first prepared the λ phage strain LIK891 with 15 PvuII sites (Materials and methods) in a host carrying PvuII methyltransferase (Materials and methods). Its modification status was confirmed by its resistance to PvuII restriction both in vitro and in vivo as follows. When the phage genome DNA prepared from the purified λ preparation was reacted with PvuII, no change was observed in its gel electrophoresis pattern under a condition where unmodified phage genome DNA was completely cleaved. The PvuII-modified phage preparation did not show detectable decreases in plaque formation efficiency in a host carrying the PvuII RM system. In an E. coli mcrBC + strain, the PvuII-modified λ phage preparation showed only a 10-fold decrease in plaque formation efficiency (Figure 3a). Consistent with previous reports [36, 37], this observation indicates that McrBC cannot efficiently restrict a methylated phage genome.

    McrBC-mediated inhibition of phage growth and chromosome cleavage. (a) Phage λ titer on ER1563 (mcrBC + ) divided by its titer on ER1562 (mcrB1) is plotted for two independent experiments. (I) A λ strain with 15 PvuII sites (LIK891 see Materials and methods) (II) the same λ strain but modified by PvuII methyltransferase (III) the same λ strain with insertion of PvuII methyltransferase gene (LEF1). (b) Chromosome degradation in ER1562 (mcrB1) and ER1563 (mcrBC + ). 5 × 10 8 cells were infected with LEF1 at a multiplicity of infection of 5. At the indicated time intervals (in minutes) after infection of phage carrying the PvuII methyltransferase gene (LEF1), chromosomal DNA was prepared and subjected to pulsed-field agarose gel electrophoresis. M, λ DNA ladder.

    However, λ phage strain LEF1, which carries the PvuII methyltransferase gene, was restricted 10,000-fold (Figure 3a). This result agrees with earlier reports indicating that phages carrying a DNA methyltransferase gene could not be propagated in an mcrBC + strain of E. coli [43]. As we noted in the previous section, whether the block to propagation is due to repeated methylation of the introduced DNA and subsequent McrBC-mediated cleavage [43] or due to host genome methylation and its McrBC-mediated lethal cleavage has not been addressed.

    When we examined chromosomes of the infected cells by pulsed-field gel electrophoresis, we observed accumulation of huge linear DNA corresponding to broken chromosomes (indicated in Figure 3b in the lanes at 30 and 45 minutes after infection) and of smaller DNAs of variable size (smear in Figure 3b in the lane at 45 minutes after infection), which likely reflect chromosome degradation. Their appearance was mcrBC + -dependent (mcrB1 lanes in Figure 3b). This observation strongly suggests that M.PvuII-mediated chromosome methylation triggered chromosome cleavage by McrBC, which was followed by chromosome degradation. This, in turn, indicates that the inhibition of their multiplication (Figure 3a) is caused by host death.

    Parenthetically, we noticed a band deriving from both the mcrB - and mcrBC + strains in the middle of the same gel and another species at the lowest position from the mcrBC + cells (data not shown). From their mobility, we inferred that these bands represent the excised circular form and the cleaved linear form of e14, a defective lambdoid phage [44, 45]. Because e14 has one PvuII site, its linear form is expected to appear after McrBC-mediated cleavage [46]. Because the lambdoid phages have similar gene organization [47–49] and regulation [50], it would not be very surprising if gene expression from the incoming λ somehow led to the expression of the excision function of e14.

    McrBC-mediated cell death and chromosome degradation following induction of the DNA methyltransferase

    The above two sets of experiments strongly suggested that McrBC mediates inhibition of propagation of the PvuII DNA methyltransferase gene through lethal cleavage of methylated chromosomes. We next asked whether induction of the PvuII methyltransferase leads to chromosome methylation followed by its McrBC-mediated cleavage and cell death. Furthermore, we asked whether we could find a close correlation between these three processes: methylation, cleavage and death.

    First, we cloned the pvuIIM gene downstream of the arabinose-inducible BAD promoter [51]. We prepared host strains for this experiment based on the work of Khlebnikov et al. [52]. These authors succeeded in achieving homogeneous expression from the BAD promoter and obtained a linear increase in the expression level in response to arabinose concentration by deleting araBAD and araFGH operons and substituting the araE promoter with a constitutive promoter [52]. We introduced these mutations to construct isogenic mcrBC +/- strains (BIK18260 and BIK18261 in Table 2). At three concentrations of arabinose (0%, 0.0002%, and 0.002%) we were able to demonstrate correlation between genome methylation, genome breakage and cell death (Figure 4) as detailed below.

    Expression of PvuII methyltransferase causes chromosome methylation and mcrBC-dependent chromosome breakage and cell death. (a) Confirmation of chromosome methylation. BIK18260 (mcrB1) cells carrying pEF24 (pvuIIM under the pBAD promoter see Table 1), were grown in LB broth under antibiotic selection to the mid-exponential phase, diluted to OD600 = 0.1, and further grown in the presence of 0.002% or 0.002% arabinose (ara) to induce expression of M.PvuII. At the indicated time intervals (in minutes), chromosomal DNA was prepared, digested with PvuII endonuclease (TaKaRa Bio), and subjected to pulsed-field agarose gel electrophoresis. M, λ DNA ladder. (b) Chromosome DNA in BIK18261 (mcrBC + ) carrying pEF24 after induction of PvuII methyltransferase. (c) Ethidium-bromide fluorescence in the well was measured for the experiments in (b). (d) Loss of cell viability. The number of viable cells was monitored in duplicate in two independent experiments. Each value was divided by the value at time zero. (e) Cell shape. The cells were recovered 60 minutes after addition of a higher (0.002%) concentration of arabinose. They were stained with DAPI to visualize nucleoids and were observed by phase-contrast (left) and fluorescence (right) microscopy. The scale bar indicates 10 μm.

    Progress in genome methylation was measured, in the mcrBC-negative strain, by resistance to PvuII cleavage in vitro (Figure 4a). The cleaved band pattern shows that the rate of progress of chromosomal DNA methylation after induction correlates with the concentration of arabinose (Figure 4a). The lower (0.0002%) concentration resulted in a delay in methylation of approximately 30 minutes compared to the higher (0.002%) concentration.

    We also followed methylation of a single PvuII site on a multi-copy plasmid (pEF60 in Table 1) included in the cell. Plasmids were extracted from cells (BIK18260) harbouring pEF60 and pEF24 (inducible M.PvuII gene) and digested in vitro with PvuII and HindIII, which cuts pEF60 at a single site. Quantification of the bands showed that the PvuII site was completely methylated 30 minutes and 60 minutes after induction with 0.002% and 0.0002% arabinose, respectively (data not shown). The time to achieve 50% methylation was about 13 minutes for the higher concentration and about 38 minutes for the lower concentration. They differed by 25 minutes. Thus, the methylation observed with the plasmid agreed well with that observed with the chromosome.

    We also observed a low level of PvuII methylation of pEF60 under the repression conditions: 4.1% and 4.3% in one experiment and 5.3% and 6.0% in another 5% corresponds to 89 sites out of 1,778 PvuII sites in the chromosome of MG1655. This indicates that PvuII methyltransferase is expressed at a low level due to slight leakage from the BAD promoter. This is consistent with earlier reports on this promoter [51, 53] and the difficulty in maintaining restriction enzyme genes under this promoter in the repressed state in E. coli [54] (M Watanabe, F Khan, Y Furuta and I Kobayashi, unpublished observation).

    The induction of PvuII methyltransferase indeed caused immediate chromosome breakage as detected by pulsed-field gel electrophoresis in the mcrBC + strain (Figure 4b) but not in the mcrBC - strain (data not shown). With the higher arabinose concentration, huge linear DNA molecules (at the middle point between the well and the 485 kb marker) became prominent by 15 minutes after the induction, and then they appeared to gradually shift into smaller fragments. With the lower arabinose concentration, the huge linear DNA molecules appeared 30 minutes after the induction and decayed in the same way. The chromosome breakage observed thus correlated well with the progress of methylation in the mcrBC - strain. Quantification of the DNAs in the well, which likely represent relatively intact chromosomes, revealed that they decreased over time after induction (Figure 4c). These decreases at the different arabinose concentrations correlated well with the progress of methylation in the mcrBC - strain.

    The chromosome breakage was accompanied by a decrease in viable cell counts (colony forming units Figure 4d). The progress of death was again related to the arabinose concentration. The stronger induction led to cell death within 15 minutes, while the weaker induction allowed maintenance of viability for 30 minutes. Many cells appeared as filaments with multiple nuclei or no nucleus (Figure 4e). Inhibition of cell growth as measured in OD was also observed in the mcrBC + cells 1-2 h after induction (Figure 5a, lower left), but not in the repressed state (Figure 5a, upper left).

    Effect of recA and recBC mutations on cell growth and chromosome changes. (a) Cell growth. BIK18260 (mcrB1), BIK18261 (mcrBC + ), BIK18290 (mcrB1 ΔrecA), BIK18291 (mcrBC + ΔrecA), BIK18292 (mcrB1 ΔrecBC) and BIK18293 (mcrBC + ΔrecBC), carrying pEF24 (pSC101::pvuIIM, see Table 1), were grown in LB broth with 0.2% glucose and selective antibiotics to exponential phase, diluted to OD600 = 0.1 and further grown with or without 0.0002% arabinose. OD600 was monitored at the indicated time intervals after addition of arabinose. Each value was divided by the value at time zero. (b) Chromosomes in uninduced cells. BIK18261 (mcrBC + ), BIK18291 (mcrBC + ΔrecA), and BIK18293 (mcrBC + ΔrecBC), and their derivatives carrying pEF24 (pSC101::pvuIIM) were grown in LB broth with 0.2% glucose and selective antibiotics to exponential phase. Chromosomal DNA was prepared and subjected to pulsed-field agarose gel electrophoresis. M, λ DNA ladder. (c) Chromosomes after induction. Chromosome DNA in BIK18261 (mcrBC + ), BIK18291 (mcrBC + ΔrecA), and BIK18293 (mcrBC + ΔrecBC), carrying pEF24 (pSC101::pvuIIM) after induction of PvuII methyltransferase with 0.002% or 0.0002% arabinose. At the indicated time intervals after induction, chromosomal DNA was prepared and subjected to pulsed-field agarose gel electrophoresis. M1, λ DNA ladder M2, λ DNA cut with HindIII.

    These results demonstrate a correlation between genome methylation, chromosome breakage, and cell death upon induction of PvuII methyltransferase. They strongly suggest that chromosomal sites methylated by PvuII methyltransferase are cleaved by McrBC and that this cleavage leads to cell death.

    Effect of mutations in DNA-related genes

    If the chromosomal sites methylated by PvuII methyltransferase are cleaved by McrBC and this cleavage leads to cell death, mutations in enzymes involved in DNA-related processes might affect these processes. We examined cell growth and chromosome changes in several mutants altered in DNA metabolism in a variety of ways.

    RecBCD enzyme is involved in exonucleolytic degradation of DNA from a double-stranded break and generates a recombinogenic single-stranded DNA end [55]. When bound to this single-stranded DNA generated by RecBCD or other enzymes, RecA protein initiates homologous pairing for recombination repair. RecA bound to single-stranded DNA also induces SOS genes through cleavage of their LexA repressor [56]. If RecA and RecBCD are involved in processing and repair of the McrBC-mediated chromosome breakage, their removal might affect cell survival and chromosome processing.

    Mutational removal of the host RecBCD/RecA exonuclease/recombinase machinery affected growth not only in the induced state but also in the repressed state (Figure 5a). A likely explanation for the uninduced state is chromosome methylation by slight expression of PvuII methyltansferase (see above). We analyzed chromosomes by pulsed-field gel electrophoresis in strain pairs with and without the PBAD-pvuIIM plasmid in the mcrBC + background. Our results shown in Figure 5b clearly indicate plasmid-dependent degradation (smear) in the recBC mutant and plasmid-dependent increase of huge linear DNAs (the thick band in the midpoint between the well and the 485 kb marker) in the recA mutant. These results strongly suggest that partial chromosome methylation led to McrBC-mediated chromosome breakage and that RecBCD/RecA machinery repairs this breakage. The defects in the repair of the McrBC-mediated chromosome breakage are likely the cause of the delayed growth of the recA and recBC mutants (Figure 5a).

    When the methyltransferase is induced, the RecBC/RecA mediated break repair presumably delays growth arrest (Figure 5a). The recA or recBC mutations slightly affected the loss of cell viability 30 minutes after the induction of methyltransferase (Table 3). However, the final viability level on exposure of the genome to methylation was similar to that in the rec + strain (data not shown).

    The chromosomes in these mutants showed changes consistent with the above growth patterns and their known properties (Figure 5c). The recBC mutant showed a large amount of huge broken chromosomes in the uninduced state these remained abundant as long as 60 minutes after induction. In the lower area, which corresponds to smaller broken chromosomes, many discrete bands are visible in the recBC mutant. This is consistent with the process in which the chromosomes broken by McrBC endonuclease were further degraded by RecBCD exonuclease. The recA mutant, unlike the rec + strain, showed more of the huge broken chromosomes even in the uninduced state. In the rec + strain, this species became prominent only 15 minutes after induction and disappeared. In the recA mutant, it remained abundant for 30 minutes but started decreasing by 45 minutes after induction. The amount of smaller broken chromosomes in the recA strain was less than that in the rec + strain, presumably due to degradation by RecBCD enzyme. No discrete bands are visible in the recA mutant, which is consistent with rapid and extensive DNA degradation by RecBCD enzyme. Discrete bands are seen in the rec + strain but they are not so many as in the recBC mutant.

    These electrophoresis patterns are consistent with the steps of McrBC-mediated chromosomal breakage, RecBCD-mediated exonucleolytic degradation from the break, and RecA-mediated homologous pairing for repair. The RecBCD/RecA-mediated repair was also found for post-segregational killing by a type II RM system [28]. From the results presented in Figure 5 and Table 3, we inferred that the RecBCD/RecA-mediated recombination repair can counteract McrBC's lethal action to some extent at a low methylation level. However, chromosome repair by them appears unable to contribute to cell survival when the genome methylation and the McrBC-mediated cleavage become extensive. This is similar to the chromosome cleavage by a mutant EcoRI enzyme [57, 58].

    The RecA/RecBCD function is also involved in the SOS response as mentioned. The cell filamentation was not observed in a recA deletion strain (data not shown). This indicates that the cell filamentation we observed represents an SOS response. In order to assess the effects of the SOS response on McrBC-mediated growth inhibition and cell death, we examined SOS-related mutants (Figure 6 and Table 3). Among these, the lexA(Ind - ) mutant is defective in SOS induction, the lexA(Def) mutant is constitutive for SOS induction, and the mutS mutant shows less background DNA breaks under some genetic backgrounds [59].

    Effect of SOS-related mutations on cell growth. BIK18262 (mcrB1 mutS), BIK18264 (mcrBC + mutS), BIK18271 (mcrB1 lexA(Ind - )), BIK18276 (mcrBC + lexA(Ind - )), BIK18278 (mcrB1 lexA(Def)), BIK18280 (mcrBC + lexA(Def)), carrying pEF24 (pSC101::pvuIIM see Table 1), were grown in LB broth with 0.2% glucose and selective antibiotics to exponential phase, diluted to OD600 = 0.1 and further grown with or without 0.0002% arabinose. OD600 was monitored at the indicated time intervals after addition of arabinose. Each value was divided by the value at time zero.

    These mutants showed McrBC-dependent growth inhibition when M.PvuII was induced, but not in the repressed state (Figure 6). McrBC-mediated inhibition observed in the lexA(Ind - ) mutant was stronger than that in the rec + strain but not so strong as in the recA strain (Figure 5a). A simple interpretation of this result is that the defect in repair in the recA-negative mutant cannot be entirely attributed to the absence of the SOS response. In other words, RecA is likely to play a direct role, presumably, in recombination repair. The lexA(Ind - ) strain also showed severe loss of cell viability 30 minutes after induction (Table 3). The results with lexA(Def) are difficult to interpret because the lexA(Def) mcrB1 strain showed slow growth. It is known that lexA(Def) mutation delays growth even in the sulA-negative background [60]. This effect could be exaggerated with McrBC-mediated chromosome breakage upon genome methylation. The mutS mutant was indistinguishable from the rec + (mutS + ) strain in these measurements. From these results, we inferred that the SOS response and RecA/RecBCD-mediated DNA recombination/repair both affect cell death/survival upon McrBC action on the methylated genome. The repair systems, however, cannot block cell death upon extensive chromosome methylation and cleavage. These observations are consistent with our hypothesis that chromosome methylation leads to its McrBC-mediated lethal cleavage.

    Generality and specificity of McrBC action against DNA methyltransferases

    In order to investigate the generality and specificity of McrBC-mediated cell death with regard to DNA methyltransferase specificity, we expressed McrBC in a cell carrying one of several methyltransferases with different specificities. First, mcrBC of E. coli was placed under the PBAD promoter (pEF46 in Table 1). As expected, McrBC induction in a cell harboring another plasmid encoding M.PvuII (CAG m4 CTG) led to cell death in the colony formation assay (Figure 7). McrBC induction also led to cell death with M.SinI (GGW m5 CC) and M.MspI ( m5 CCGG) (Figure 7) but not with M.SsoII (C m5 CNGG) (data not shown). These results are consistent with the R m C sequence specificity of McrBC observed in vitro [33]. Our interpretation is that McrBC has the potential to act as a defense system against many DNA methyltransferases of an appropriate specificity.

    McrBC-mediated cell death with DNA methyltransferases. Cells (BIK18308) harboring pEF46 (PBAD-mcrBC see Materials and Methods) and pEF43 (M.PvuII), pSI4 (M.SinI), pNW106RM2-3 (M.MspI), or pBAD30 (vector) were streaked on LB agar plate containing 30 μg/ml chloramphenicol and 25 μg/ml ampicillin, and 0.2% glucose or 0.2% arabinose. Plates were incubated overnight at 37°C.

    Molecular evolutionary analyses of McrB and McrC reveal their frequent loss and horizontal transfer between distantly related genomes

    The above experimental results provide an answer to the question we first formulated. It is very likely that McrBC cleaves host chromosomes and causes cell death upon genome methylation and that this cell death inhibits propagation of the methyltransferase gene (Figure 1b). McrBC was also demonstrated to severely restrict bacteriophages carrying hydroxymethylated C in place of C in their genomes [34, 35, 61, 62]. Which of these actions of McrBC has been providing selective advantage for their spread and maintenance during evolution?

    In order to address this question, we focused on the similarity of McrBC with type II RM systems in the action of host killing by chromosome cleavage. As illustrated in Figure 1a, when a type II RM gene complex is replaced by a competitor genetic element, its product restriction enzyme will cleave host chromosomes in which methylation decreases and kill the host (Figure 1a) [22]. This leads to survival of cells retaining the RM gene complex but not its competitor. The McrBC system may likewise contribute to exclusion of epigenetic methylation systems (Figure 1b). A contrast between them is that McrBC action follows gain of methylation, as opposed to loss of methylation.

    The potential for host killing by type II RM systems indicates their relative independence from the host. They act as a unit of selection and, in this regard, they might be similar to viral genomes, transposons and other selfish mobile elements. Indeed, there are now many lines of evidence for the mobility of type II RM systems [21]. These include molecular evolutionary evidence for their extensive horizontal transfer between distantly related prokaryotes, carriage by mobile elements such as plasmids and linkage with mobility-related genes. Likely due to this mobility, in addition to the ability to cut incoming DNAs and to fight against competing elements by host killing, type II RM systems are widespread throughout Prokaryota. They are often lost from a genome by various mutations [21]. They are quite diversified in sequence recognition because of frequency-dependent selection in defense against incoming DNAs [63] and/or because of mutual competition for recognition sequence in host killing [18]. We asked whether McrBC homologs show similar properties. If they do so, we might take it as evidence supporting the hypothesis that McrBCs have evolved for their ability to kill the host cell in competition with genome methylation systems and behave as selfish mobile elements.

    In order to address these points and evaluate the above two hypotheses for McrBC, we examined its evolutionary history. Using the sequence of McrB and McrC from E. coli as queries for PSI-BLAST [64] searches, we identified 199 homologous McrBC-like systems, typically comprising operons with an mcrB-like gene followed by a mcrC-like gene (see also below). These homologs of the McrBC system are widely distributed in Bacteria and Archaea (Table S1 in Additional data file 1), like, for example, type I or type II RM systems [17]. If mcrBC homologs show a very narrow distribution and this correlates with distribution of phages with hydroxymethyl C, the phage defense hypothesis might be favored. We address these issues in the Discussion.

    Phylogenetic trees calculated from multiple sequence alignments of McrB and McrC sequences (Materials and methods) reveal very similar topologies, suggesting strong co-evolution of these two proteins (Figure S1 in Additional data file 2). Nine bootstrap-supported branches reveal relationships between sequences from different taxons, indicating a very high probability of distant horizontal gene transfer events, which is also a feature of evolution of type II RM systems [15, 65]. In the aforementioned cases, McrB and McrC appear to have experienced joint horizontal transfer.

    The mcrBC gene complex in E. coli K12 was suggested to have been acquired recently [61], which is confirmed by our phylogenetic analysis: McrB and McrC from E. coli K12 are not found in a branch specific to Proteobacteria (top part of the tree in Figure S1 in Additional data file 2), but in a branch that also includes Acidobacteria bacterium Blin 345 (the closest homolog of E. coli McrBC), Firmicutes, and Actinobacteria. In general, McrBC subunits from taxons such as Proteobacteria, Actinobacteria, or Firmicutes form numerous intermixed branches in the tree, suggesting multiple horizontal gene transfers followed by vertical dissemination among diverging species and strains. One example of a branch of functionally similar enzymes from completely different taxons is provided by the family of unusual type II RM systems related to McrBC (including LlaI [66], BsuMI [67], LlaJI [68] and their experimentally uncharacterized homologs) that cleave unmethylated DNA and are accompanied by a pair of type IIS DNA methyltransferases to protect against the cleavage of their self-DNA (labelled type II R-like subfamily in Figure S1 in Additional data file 2).

    Another feature revealed by the phylogenetic trees is the presence of two strongly diverged subfamilies of McrBC-like systems, one comprising known McrBC (for example, the one from E. coli K12) and McrB-like systems (for example, the aforementioned type II enzymes), and the other comprising solely uncharacterized McrBC-like homologs of unknown function, with the McrC-like component defined as uncharacterized protein family DUF524. It is interesting that members of these two subfamilies show nearly perfectly complementary phylogentic distribution, that is, despite their presence in similar taxons, they do not co-occur in one genome (Table S2 in Additional data file 3 and Table S1 in Additional data file 1), which probably reflects some degree of their mutual incompatibility.

    The few events of distant horizontal transfer indicated on the phylogenetic trees correspond only to those cases where an McrB (and/or McrC) homolog from one taxon is found to be embedded in a branch comprising a different taxon (for example, Deinococcus within Gammaproteobacteria) and where this branch has bootstrap support >50%. This is a very conservative estimation of horizontal gene transfer events. The trees reveal many other cases of branches with mixed taxons, but their bootstrap support is <50%, indicating lack of statistical support for the local tree topology. When we compared the McrB and McrC trees with the 16S rRNA trees calculated for the same set of species (Figure S2 in Additional data file 4), we found numerous disagreements in deep branches, and agreement only in short branches that connect closely related species. This analysis suggests that McrBC systems have been transmitted horizontally numerous times, but of course they have been also inherited vertically by closely related groups of organisms radiating from their common ancestor (for example, by strains of the same species, such as Streptococcus pneumoniae, Campylobacter jejuni, or Yersinia pestis). However, it is very difficult to quantify the rate of distant horizontal transfer by analyzing a tree with a highly variable bootstrap support for different nodes therefore, we resorted to an independent strategy.

    Gojobori and coworkers [69] have published analysis of 116 completely sequenced prokaryotic genomes, in which they calculated an index of potential distant horizontal transfer for all genes, by comparing the frequency of 'words' of pentanucleotide length within each gene with the average word frequency of the entire genome. We have obtained an updated data set for 165 genomes from Dr Nakamura and Dr Gojobori (personal communication). Among these genomes, 29 contain both McrB and McrC homologs (D. radiodurans contains one additional McrB homolog). We have analyzed the horizontal transfer index of all genes encoding McrB and McrC homologs and found that 9 McrB-homologous genes (9/30 = 30%) and 10 McrC-homologous genes (10/29 = 35%) exhibit word frequencies that indicate significant likelihood of distant horizontal gene transfer. Thus, in the sample of McrBC systems, for which data are available, approximately one-third appears to have been derived by a recent horizontal gene transfer event from a distantly related group. For the same set of genomes, we also carried out analysis of the horizontal transfer index of genes from two reference 'house-keeping' protein families: RecA and RpoB. We found no members of RecA or RpoB genes in this sample to be predicted as recently transferred.

    We found that the McrBC gene complex tends to be lost quite frequently, as no higher-order taxon is found in which all completely sequenced genomes possess this system. Among 567 completely sequenced genomes in which we looked for McrB/C homologs, we found McrB in only 112 cases (19.8%) and McrC in 108 cases (19.0%) McrB and McrC were found together in 107 cases (18.9%). Thus, we conclude that McrBC systems are frequently transmitted by horizontal gene transfer (in addition to regular vertical transfer), but are also very frequently lost. This argues against the hypothesis that they are conserved due only to their utility for defense against phages or other parasites and favors the hypothesis they behave as selfish (host-killing) mobile elements.

    Genomic neighborhood analysis of McrBC systems suggests their mobility and linkage with genome methylation systems

    Type II RM gene complexes are often found on mobile elements such as plasmids, phages, integrons and genomic islands [21]. In accord, they are often linked with mobility-related genes such as transposase homologs and integrase homologs. We examined the neighbourhoods of mcrBC homologs expecting to find similar genes.

    Genomic neighbourhood analysis (Table S2 in Additional data file 3 see Table S1 in Additional data file 1 for the complete data set) revealed that McrB and McrC are tightly linked to each other, suggesting their structure as a single operon. They are frequently associated with homologs of integrases and transposases (Table S2 in Additional data file 3 and Table S1 in Additional data file 1). Several McrBC homologs clearly occur as an insert in an RM gene complex (Figure 8). In addition, eight McrBC-like systems were found on a plasmid (Table S1 in Additional data file 1). These three lines of evidence indicate potential mobility of the mcrBC unit. The mcrBC homologs were often linked with RM systems or just DNA methyltransferases (Table S2 in Additional data file 3), as first noted for E. coli [70]. The implication of this finding is discussed below.

    mcrBC-like homologs apparently inserted into an RM gene complex. Open reading frame names indicate enzyme names (REBASE) or locus tags (GenBank).

    Some genomes, such as the Deinococcus radiodurans R1 genome, contain two mcrBC homologs, sometimes one on a plasmid and the other in the chromosome. Alignment of these pairs of McrB homologs found in the same genome revealed that their amino acid sequences often vary in the amino-terminal region, which is involved in DNA binding [46], suggesting evolutionary shifts in DNA sequence specificity (Figure 9). This parallels the diversity in sequence recognition of type II restriction and modification enzymes.

    Dot-plot comparison of intragenomic mcrB paralogs. Amino acid sequences of a pair of mcrB paralogs within one genome were plotted against each other.

    To investigate the relationship between the diversity of the McrB amino-terminal region and sequence recognition, several McrBC homologs, STOMcrBC (NP_377078.1) and STOMcrBC2 (NP_377080.1) from Sulforobus tokodaii str. 7, TKOMcrBC (YP_183208.1) and TKOMcrBC2 (YP_183422.1) from Thermococcus kodakaraensis KOD1, and DraMcrBC (NP_051672.1) from D. radiodurans R1, were amplified from genome DNA and cloned into pBAD30 [51]. These mcrBC homologs did not cause cell death in E. coli at 37°C in the presence of arabinose in a cell harboring either of the four DNA methyltransferase genes, M.PvuII (CAG m4 CTG), M.SinI (GGW m5 CC), M.MspI ( m5 CCGG), or M.SsoII (C m5 CWGG) (data not shown). EcoKMcrBC from E. coli caused cell death sensing genome methylation by M.SinI (GGW m5 CC) and M.MspI ( m5 CCGG) under the same condition (Figure 7). Therefore, we were unable to link these homologs with the biology of the organisms.


    Many of the α-proteobacteria establish long-term, often chronic, interactions with higher eukaryotes. These interactions range from pericellular colonization through facultative intracellular multiplication to obligate intracellular lifestyles. A common feature in this wide range of interactions is modulation of host-cell proliferation, which sometimes leads to the formation of tumour-like structures in which the bacteria can grow. Comparative genome analyses reveal genome reduction by gene loss in the intracellular α-proteobacterial lineages, and genome expansion by gene duplication and horizontal gene transfer in the free-living species. In this review, we discuss α-proteobacterial genome evolution and highlight strategies and mechanisms used by these bacteria to infect and multiply in eukaryotic cells.

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