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Proteobacteria

Phylum Proteobacteria (Purple Bacteria & relatives)

Most of the familiar Gram-negative Bacteria are members of the Proteobacteria. The members of this very successful phylum predominate many environments. Most of the familiar Gram-negative Bacteria are members of the phylum. They have a very broad range of phenotypes scattered around the phylogenetic tree. This implies that these organisms can change phenotype readily, at least in terms of evolutionary history; for this reason they have been named after the shape-shifting sea god Proteus. Unlike most of the other bacterial phyla, there is no phenotypic trait that units the Proteobacteria. The purple phototrophic Bacteria are members of this group, and so this phylum is also sometimes referred to as the Purple Bacteria and relatives. In addition to purple phototrophs, members of this phylum include many heterotrophs (including some important symbionts and pathogens) and key players in the sulfur and nitrogen cycles.

Because this is such a broad phylum, with thousands of described species, the Proteobacteria are usually divided into classes, which are given the generic labels α-, β-, γ-, δ- and ε-proteobacteria. Perhaps this generic labeling has persisted because even within each of these classes there is a range of phenotypes; heterotrophs, phototrophs, and chemoautotrophs of various kinds, without any unifying themes.

Given the incredible diversity of organisms in the phylum Proteobacteria, we will only be able to touch on a few examples.

Class α-proteobacteria

• Class α-proteobacteria
  • Order Caulobacterales
    • Family Caulobacteraceae (e.g. Caulobacter)
  • Order Parvularculales
    • Family Parvularculaceae (Parvularcula)
  • Order Rhodobacterales
    • Family Rhodobacteraceae (e.g. Rhodobacter, Paracoccus, Roseobacter)
  • Order Rhodospirillales
    • Family Acetobacteraceae (e.g. Acetobacter, Gluconobacter, Stella)
    • Family Rhodospirillaceae (e.g. Rhodospirillum, Magnetospirillum)
  • Order Rhizobiales
    • Family Aurantimonadaceae (e.g. Aurantimonas)
    • Family Bartonellaceae (e.g. Bartonella, Rochalimaea)
    • Family Beijerinckiaceae (e.g. Beijerinckia, Methylocella)
    • Family Bradyrhizobiaceae (e.g. Bradyrhizobium, Rhodopseudomonas)
    • Family Brucellaceae (e.g. Brucella, Ochrobactrum)
    • Family Hyphomicrobiaceae (e.g. Hyphomicrobium, Ancylobacter)
    • Family Methylobacteriaceae (e.g. Methylobacter, Microvirga, Meganema)
    • Family Methylocystaceae (e.g. Methyloyctis, Pleomorphomonas)
    • Family Phyllobacteriaceae (e.g. Phyllobacterium, Mesorhizobium)
    • Family Rhizobiaceae (e.g. Rhizobium, Agrobacterium)
    • Family Rhodobiaceae (e.g. Rhodobium, Roseospirillum)
  • Order Rickettsiales
    • Family Anaplasmataceae (e.g. Anaplasma, Ehrlichia, Wolbachia)
    • Family Holosporaceae (Holospora)
    • Family Rickettsiaceae (e.g. Rickettsia, Orientia)
  • Order Sphingomonadales
    • Family Sphingomonadaceae (e.g. Sphingomonas, Erythrobacter)
      

General characteristics of the α-proteobacteria

Diversity
The α-proteobacteria are a large group of organism encompassing 7 Orders, 20 Families and more than 200 genera. Also in this group are the mitochondria of eukaryotes, probably as relatives of the Rickettsiales although this is obscured by the very high evolutionary rates in the gene sequences of the mitochondria.

Metabolism
Most of the purple non-sulfur phototrophic Bacteria are members of the α-proteobacteria, but there are also a wide range of heterotrophic species as well, including some important pathogens. Also in this Class are autotrophic methane oxidizers (methylotrophs) and a many organisms that are capable of nitrogen fixation, usually as symbionts of plant hosts. Autotrophic members of this Class fix CO2 via the Calvin cycle. The Sphingomonads lack lipopolysaccharide (LPS) in their cell envelopes, having instead glycosphingolipids similar to those found in the central nervous systems of animals.

Morphology
Although most of the members of this Class are generic rods and cocci, and a few spirilla, many of the appendaged Bacteria are members of this group. These appendages are extensions of the cell, surrounded by the cells envelop, unlike the stalks of the Planctomycetes in which these are fibrous extracellular structures. Appendages can be single stalks such as in Caulobacter, or multiple “arms” as in Ancalomicrobium. In the Hyphomicrobia, including the purple phototroph Rhodomicrobium, reproduction is by budding from the ends of appendages, often resulting in chains and networks of cells connected by thin bridging appendages.

Habitat
Members of this class are abundant in nearly all habitats, and are predominant members of both aerobic and anaerobic aquatic environments.

Purple non-sulfur phototrophs

With the exception of the genus Rhodocyclus and a couple of close relatives, all purple non-sulfur Bacteria are members of the α-proteobacteria. These organisms carry out anoxygenic photosynthesis using a single photosystem, i.e. cyclic photophosphorylation. Although all can grow photoheterotrophically, using light for energy and organic compounds for carbon growth requirements, most are also capable of photoautotrophy using hydrogen or reduced sulfur compounds as a source of reducing power for carbon fixation; this is captured in the form of NADH as the product of reverse electron flow, and is used to fix carbon via the Calvin cycle. A few can grow fermentatively producing organic acids, CO2 and hydrogen, or by anaerobic respiration using nitrate or nitrite as terminal electron acceptors. Most are capable of nitrogen fixation. Photosynthesis takes place in internal membranes, which can take the form of vesicles lining the cytoplasmic membrane, flattened stacks resembling thylakoids, or concentric lamellae lining the cytoplasmic membrane.

Example: Rhodomicrobium vannielii

Rhodomicrobium is a commonly-isolated purple phototroph, producing deep red growth in liquid culture or colonies. It is an appendaged ovoid or rod-shaped cell that reproduces by budding from the ends of appendages. It prefers to grow photoheterotrophically, and is capable of utilizing a wide range of organics, but in the absence of these organic compounds it grows photoautotrophically using sulfide or hydrogen as the source of reducing power for carbon fixation. Thiosulfate, not elemental sulfur nor sulfate, is the product of sulfide oxidation. Unlike some other purple non-sulfur Bacteria, R. vannielii is not capable of nitrogen fixation. It is also unique amongst these organisms in having peritrichous flagella and in forming moderately heat-resistant resting cysts (exospores). Photosynthetic membranes are lamellar.

Appendaged Bacteria

The appendaged, or “prosthecate” α-proteobacteria contain cytoplasmic extensions. Most are aerobic heterotrophs, and live in oligotrophic environments. If only a single appendage is present, it is referred to as a stalk. These stalks are used to attach to surfaces, with terminal holdfasts often containing powerful adhesives. In those with multiple appendages, these apparently serve to increase the surface area of the cells, and they may also make them resistant to grazing by some protists. Most divide by binary fission, but a few divide by budding from the ends of appendages (e.g. Hyphomicrobium).

Some stalked α-proteobacteria are dimorphic; reproducing by binary fission in which one offspring resembles the original non-motile stalked “mother” cell, but the other offspring is flagellated and lacks a stalk. These motile “swarmer” cells disperse in the environment, attach to a surface, and develop into sessile stalked cells. This dispersal prevents the accumulation of non-motile cells in one place in which they would compete with each other for resources in their usual oligotrophic environment; this is the same reason most sessile reef animals have planktonic larvae.

Example: Caulobacter crescentus

C. crescentus is a well-studied dimorphic appendaged bacterium. Cells are vibriod or fusiform. Mature cells have a single thin terminal stalk. Division is by binary fission; before division is complete, a single flagellum is created at the pole opposite the stalk. Although the mother and swarmer cells produced are approximately the same size, the DNA of the swarmer cell is condensed and transcriptionally inactive, and metabolism in the swarmer is much reduced compared to the mother cell. When the swarmer cell comes in contact with a solid substrate, it adheres, loses its flagellum, and develops a stalk (with a terminal holdfast) from the same end of the cell that previously had the flagellum. Caulobacter is ubiquitous in aquatic samples, and is most readily observed attached to diatoms and eukaryotic algae. Caulobacter, and other appendaged Bacteria, are readily isolated from the surfaces inside laboratory distilled water containers.

Nitrogen-fixing plant symbionts

These organisms, known as the Rhizobia, form intimate symbioses with leguminous plants. The Bacteria enter the plant via the root hairs, enter the body of the root through an infection thread, and induce the formation of root nodules. In these nodules, the bacterial cells reproduce, then develop into the symbiotic form (“bacteroids”), that fix atmospheric nitrogen both for the bacterial symbionts and for the host plant. These bacteroids are terminally differentiated; they cannot revert to vegitative bacteria, nor can they reproduce. The host provides the bacteroids with nutrients and vitamins for grow and metabolism in return. The growth of these leguminous plants, with their nitrogen-fixing rhizobia, is commonly used to replenish impoverished soil.

As described previously in the discussion of nitrogen fixation by cyanobacteria, nitrogenases (the enzymes that reduce N2 to NH4+) are strongly inhibited by oxygen. Rhizobia, however, are obligate aerobes. In culture, Rhizobia can be grown and fix nitrogen microaerophilically. In nodules, the bacteroids are provided with plenty of oxygen bound to a legume-hemoglobin, leghemoglobin, that is readily available for respiration but maintains a very low concentration of free oxygen that would inhibit nitrogenase.

The production of effective (nitrogen-fixing) nodules is a complex process requiring a matching pair of compatible host and bacterial symbiont. Mis-matched pairs of host:symbionts often can produce ineffective nodules that fix little or no nitrogen.

When the host plant dies, the small numbers of vegetative (non-bacteroid) rhizobia escape from the decaying nodule and persist in the soil, capable of infecting emerging host plants.

Example: Rhizobium etli

This species, previously known as Rhizobium leguminosarum biovar phaseoli type I, is specific for the host Phaseolus vulgaris, the Latin American common bean. The genome of R. etli is comprised of a single large chromosome (4.38Mbp) and 6 smaller chromosomes ranging from 0.18 to 0.64Mbp. Because the smaller chromosomes contain mostly non-essential genes, they would usually be considered plasmids, but they contain a wide range of important genes, including genes required for normal cell cycling and the nod genes required for their normal symbiotic life cycle. The largest of these so-called plasmids are as big as the entire genomes of some obligately parasitic Bacteria!

Obligate intracellular parasites

These include a wide range of obligately intracellular symbionts or parasites of eukaryotes, especially animals. Best known are the insect symbionts (e.g. Wolbachia) and insect-borne human and mammal pathogens (e.g. Rickettsia). Also a member of this group phylogenetically, Bartonella (previously Rochalimaea) is an obligate parasite, but lives on the outside surface of the host cell rather than intracellularly. These organisms are often compared to viruses, but although small and obligately intracellular parasites, they are otherwise typically bacterial.

Also members of this group are the mitochondria of eukaryotes. These seem to be specifically related to Rickettsia, the causative agents of spotted fevers and typhus. Both groups lack enzymes for glycolysis (this being carried out in the cytoplasm of eukaryotes), but contain the enzymes of the complete TCA cycle and electron transport. Amino acids and nucleotides precursors are provided by the host in either case. The sequences encoding these proteins in Rickettsia resemble those of mitochondria, even in cases where these genes now reside in the nuclear genome rather than in the mitochondrial genome.

Example: Wolbachia pipientis

W. pipientis is the only formally described species of this genus. Wolbachia is a very common intracellular symbiont of arthropods and nematodes. A majority of insect species seem to be susceptible to infection, and perhaps 15-20% of individuals are infected. Although infection with Wolbachia is not associated with outright disease, it does cause a range of phenotypes in the reproductive biology of the host. This is because infected females transmit the symbiont directly to their eggs, but infected males do not transmit the symbionts to their offspring via infected sperm. As a result, in an effort to maximize the number of infected offspring their host generates, the symbiont manipulates the host in favor of producing females at the expense of males. Often this means inducing parthenogenesis, feminization (causing genetically male eggs to develop into females), or embryonic lethality in males (son-killing). In insects in which infected males are viable, they can usually only successfully fertilize the eggs from females infected with the same strain of Wolbachia, resulting in the phenomenon of “cytoplasmic incompatibility”; this seems to be mediated by imprinting of the hosts chromosomes in both sperm and eggs. This can create a reproductive barrier favoring host speciation in the absence of geographic isolation.

Class β-proteobacteria

• Class β-proteobacteria
  • Order Burkholderiales
    • Family Alcaligenaceae (e.g. Alcaligenes, Bordetella, Achromobacter)
    • Family Burkholderiaceae (e.g. Burkholderia, Ralstonia)
    • Family Comamonadaceae (e.g. Comamonas, Acidovorax)
    • Family Oxalobacteraceae (e.g. Oxalobacter, Janthinobacter)
  • Order Hydrogenophilaceae
    • Family Hydrogenophilaceae (e.g. Hydrogenophilus, Thiobacillus)
  • Order Methylophilales
    • Family Methylophilaceae (e.g.Methylophilus, Methyobacillus)
  • Order Neisseriales
    • Family Neisseriaceae (e.g. Neisseria, Aquaspirillum, Vitreoscilla)
  • Order Nitrosomonadales
    • Family Gallionella (Gallionella)
    • Family Nitrosomonadaceae (e.g. Nitrosomonas, Nitrospira)
    • Family Spirillaceae (Spirillum)
  • Order Procabacterales
    • Famly Procabacteraceae (Procabacter)
  • Order Rhodocyclales
    • Family Rhodocylaceae (e.g. Rhodocyclus, Azoarcus, Zoogloea)
      

About this Class

Diversity
Although less diverse phylognetically, and perhaps less abundant in the environment, than either the α- or γ-proteobacteria, the β-proteobacteria nevertheless are a diverse and abundant Class of Bacteria, consisting of at least 125 characterized genera.

Included in this Class are a number of organisms that were previously considered to be members of the genus Pseudomonas. This genus is now reserved for organisms specifically related to P. aeruginosa and P. fluorescens; other phylogenetic groups that had been lumped into this genus on the basis of generic phenotypic criteria have been divided into a number of new genera and reclassified on the basis of their phylogenetic relationships.

Metabolism
Like the other classes of proteobacteria, the β-protobacteria are very diverse metabolically and phenotypically. Most of these organisms are either heterotrophs (including some important pathogens, although mostly opportunists) or chemolithoautotrophs. They are generally aerobic or facultatively anaerobic.

Heterotrophic members of this Class are able to utilize a wide range of substrates for growth, including compounds important in waste management, such as phenol and lignin. Lithotrophic members are key players in the nitrogen cycle, primarily in nitrification. Many are also sulfur, ferrous iron, or manganese oxidizers, and those that grow autotrophically use the Calvin cycle to fix CO2. Also in this Class are the methylotrophs, methane oxidizers.

Although the bulk of the purple non-sulfur phototrophs are members of the α-proteobacteria, one main genus (Rhodocyclus) and a few less well-known genera (Rubrivivax, Roseateles) are members of the β-proteobacteria. Morphologically, Rhodocyclus are very tightly wound short helices, resembling old-fashioned lockwashers.

Morphology
Although typically rods and cocci, there are a number of conspicuous spirilla in this Class, and the non-cyanobacterial filamentous sheathed Bacteria. Filamentous iron- and manganese-oxidizing organisms typically become coated with granules of insoluble metal salts. Simonsiella and its relatives are gliding flattened multicellular filaments that inhabit the oral epithethium of mammals, often in great numbers.

Habitat
These organisms are abundant in most environments, particularly in organic-rich soils, sediments, wastewater and eutrophic aquatic systems. Also common are those found in association with plant and animal surfaces.

Heterotrophs and pathogens

The β-proteobacteria contain numerous heterotrophic species that are common in the environment, and are capable of utilizing a wide range of organic compounds. Most of these heterotrophs are aerobic rod-shaped organisms, and many are at least opportunistic pathogens of plants and animals. The most important animal pathogens include Bordetella, Burkholderia, and Neisseria.

Example: Ralstonia solanacearum

R. solanacearum is an important plant pathogen, causing “Southern bacterial wilt” in a wide range of crop plants worldwide, including tobacco, potato (Brown rot), tomato, pepper and bananas (Moko disease). Species now in the genus Ralstonia were previously members of the genus Pseudomonas, and are obligately aerobic motile rods. The pathogen enters the plant through the root hairs, and grows and is transported throughout the plant in the xylem. The organism grows to such high numbers in the plant that one of the standard diagnostic tests is to touch the cut end of an infected stem to a container of water; if the infection is R. solanacearum, cells and exopolysaccharide can been easily seen as a milky stream flowing out of the xylem. R. solanacearum can overwinter in the soil or water, or persist for even longer periods of time. The genome of R. solanacearum is in two chromosomes, a 3.6Mbp circle containing most of the essential genes, and a 2.1Mbp “megaplasmid” that also contains essential genes and genes required for pathogenicity.

Chemolithoautotrophs

Most of the chemolithoautotrophs amongst the β-proteobacteria fall into four categories; sulfur oxidizers (that can sometimes oxidize metal ions as well), methylotrophs, ammonia oxidizers, and hydrogen oxidizers. None of these phenotypes is unique to the β-proteobacteria; all are also found at least the α- and β-proteobacteria. Many of these organisms are obligate autotrophs. The Calvin cycle is used for CO2 fixation.

The most prevalent group of chemoautotrophs in the β-proteobacteria are the sulfur oxidizers. These organisms use reduced sulfur compounds (sulfide, thiosulfate, thiocyanate, elemental sulfur) as the reductant for electron transport; oxygen is the common terminal electron acceptor, although many can use nitrate or nitrite as alternative terminal electron acceptors. Sulfuric acid is the product of sulfur oxidation, although some accumulate elemental sulfur as long a more reduced sulfur compounds are available. These organisms are ubiquitous in environments at the interface between aerobic and anaerobic zones, where sulfides and oxygen coexist. Many are capable of iron oxidation (ferrous to ferric); this, combined with their production of sulfuric acid from sulfur oxidation, makes them important contributors to the corrosion of plumbing. Sulfur oxidizers are used routinely in the mining process to extract metals from ores by leaching.

Example: Thiobacillus thioparus

[ photo ]

T. thioparus is an obligate chemolithotroph, oxidizing reduced sulfur compounds using oxygen or nitrate as the terminal electron acceptor. Nitrate is reduced to nitrite. This rod-shaped motile organism is an neutrophile, in contrast to the acidophilic γ-proteobacteria Athiobacillus species that were previously in this genus. Carboxysomes are present when grown under conditions of CO2 limitation; these are small proteinaceous organelles that concentrate CO2 and contain the enzymes of the Calvin cycle. Many organisms that use the Calvin cycle contain carboxysomes.

Sheathed filaments

The sheathed filamentous β-proteobacteria are commonly seen in polluted streams and wastewater. They are distinguished from other filamentous Bacteria, which are common in these environments, by their sheath, which is a tubular structure surrounding the cells of the filament. Sheathed β-proteobacteria usually “strengthen” their sheaths with a precipitate of iron hydroxide of manganese dioxide, which he organisms produce by oxidation of soluble reduced metal ions. These organisms are obligately aerobic heterotrophs; it is unlikely that energy from the oxidation of metals contributes to the energy needs of the cells. Sheaths often are surounded by a slime layer, and attached to the substrate by a terminal holdfast. In addition to attachment, the sheath protects the filaments from predation.

Example: Sphaerotilus natans

S. natans is an iron-accumulating organism, perhaps the most commonly seen member of this group. Filaments contain false branches, breaks in the sheath from which filaments can protrude. Sheaths are thin and smooth, and are brown in color because it their impregnation with iron hydroxide. Filaments can sometimes leave their sheath and create now ones, or individual cells can leave to create new filaments. Empty space in sheaths is common. Filaments are 1.2-2.5μm in diameter, and individual cells are 2-10μm in length. S. natans is commonly found in aerated wastewater, here it can be a nuisance, contributing to “bulking”, poor settling of sludge.

Class γ-proteobacteria

• Class γ-proteobacteria
  • Order Acidithiobacillales
    • Family Acidithiobacillaceae (Acidothiobacillus)
    • Family Thermithiobacillaceae (Thermithiobacillus)
  • Order Aeromonadales
    • Family Aeromonadaceae (e.g. Aeromonas, Oceanomonas)
    • Family Succinivibrionaceae (e.g. Ruminobacter, Succinivibrio)
  • Order Alteromonadales
    • Family Alteromonadaceae (e.g. Alteromonas, Glaciecola)
    • Family Colwelliaceae (Colwellia, Thalassomonas)
    • Family Ferrimonidaceae (Ferrimonas)
    • Family Idiomarinaceae (e.g. Idomarina)
    • Family Moritellaceae (Moritella)
    • Family Pseudoalteromonadaceae (Algicola, Pseudoalteromonas)
    • Family Psychromonadaceae (Psychromonas)
    • Family Shewanellaceae (Shewanella)
  • Order Cardiobacteriales
    • Family Cardiobacteriaceae (e.g. Cardiobacter)
  • Order Chromatiales
    • Family Chromatiaceae (e.g. Chromatium, Thiocapsa, Thiocystis)
    • Family Ectothiorhodospiraceae (e.g. Ectothiorhodospira, Nitrococcus)
    • Family Halothiobacillaceae (e.g. Halobacillus, Thiovirga)
  • Order Enterobacteriales
    • Family Enterobacteriaceae (e.g. Buchnera, Escherichia, Yersina)
  • Order Legionellales 
    • Family Coxiellaceae (e.g. Coxiella, Rickettsiella)
    • Family Legionellaceae (e.g. Legionella)
  • Order Methylococcales
    • Family Methylococcaceae (e.g. Methylobacter, Methylococcus)
  • Order Oceanospirillales
    • Family Alcanivoraceae (e.g. Alcanivorax)
    • Family Hahellaceae (e.g. Hhella, Halospina)
    • Family Halomonadaceae (e.g. Chromohalobacter, Halomonas)
    • Family Oceanospirillaceae (e.g. Marinomonas, Oceanospirillum)
    • Family Oleiphilaceae (Oleophilus)
    • Family Saccharospirillaceae (Saccharospirillum) 
  • Order Pasteurellales
    • Family Pasteurellaceae (e.g. Actinobacillus, Haemophilus, Pasteurella)
  • Order Pseudomonadales
    • Family Moraxellaceae (e.g. Acinetobacter, Psycrobacter, Moraxella)
    • Family Pseudomonadaceae (e.g. Azotobacter, Pseudomonas)
  • Order Thiotrichales
    • Family Francisellaceae (Francisella)
    • Family Piscirickettsiaceae (e.g. Thiomicrospira, Methylophaga)
    • Family Thiotrichaceae (e.g. Beggiatoa, Thiothrix, Thiomargita)
  • Order Salinisphaerales
    • Family Salinisphaeraceae (Salinsphaera)
  • Order Vibrionales
    
    • Family Vibrionaceae (e.g. Photobacterium, Vibrio)
  • Order Xanthomonadales
    • Family Xanthomonadaceae (e.g. Stenotrophomonas, Xanthomonas)
      

About this Class

Diversity
The γ-proteobacteria is a very large and diverse Class. As mentioned at the beginning of the Chapter, most of the familiar Gram-negative Bacteria are members of the proteobacteria, but even within this large and diverse Phylum, a large fraction of familiar Gram-negative organisms are specifically members of the Class γ-proteobacteria. This Class contains 15 orders, 35 recognized families, and about 250 genera.

Metabolism
Members of this Class span the metabolic gamut. There are obligate aerobes, facultative anaerobes, microaerophiles and obligate anaerobes; heterotrophs, chemoautotrophs and photoautotrophs; deadly pathogens, opportunistic pathogens and life-sustaining symbionts; and cryophiles, mesophiles and moderate thermophiles. Most metabolic possibilities have examples in this Class. This being said, most familiar γ-proteobacteria are heterotrophic.

Morphology
Most of the organisms in this group fall into the stereotypical bacterial morphologies and sizes; rods, cocci and some spirilla. Curved rods (vibrios) are also common, and a few are filamentous. An occasional organism will fall outside of this range, an extreme example being the sulfide and nitrate oxidizing Thiomargarita, a spherical organism with cells nearly a millimeter in diameter.

Habitat
The γ-proteobacteria are ubiquitous in nature, very often making up the largest fraction of the population. They are not known to exist in hyperthermophilic or strongly alkaline environments, but the γ-proteobacteria can be found almost anywhere except the fringes of the limits of life.

Enterics

The Enterobacteria (enterics) are probably the best studied group of Bacteria. They are easy to isolate, grow and manipulate, very common, and are important symbionts (and pathogens) of plants and animals. Enterics are mesophilic facultatively anaerobic heterotrophs, growing respiratively in aerobic conditions and fermentatively under anaerobic conditions. Many can also grow by anaerobic respiration using nitrate as the terminal electron acceptor, producing nitrite. Motility is by peritrichous flagella; a few are non-motile. Acid and gas are commonly produced from carbohydrate fermentation. They are oxidase negative but catalase positive with few exceptions.

Most or all of the enterics are at least opportunistically pathogenic, and this group contains many well-known important pathogens; Salmonella, Shigella, Escherichia, Yersina, and Klebsiella are good examples. Plant pathogens in this group are responsible for a wide range of plant wilts and blights in commercial and wild plants.

Example: Escherichia coli

Certainly the most well-known and understood of Bacteria. It is generally a commensalistic inhabitant of mammalian colon. Because it is much easier to grow than are the far more abundant obligately-anaerobic Clostridium and Bacteroides, it is routinely used as an indicator of fecal contamination of food and water. It is the standard workhorse of molecular biology because it is easy to grow, safe, and readily manipulated genetically. Nevertheless, it is an opportunistic pathogen; even otherwise benign strains can cause urinary tract infections. More virulent strains cause gastroenteritis ranging from mild to life-threatening.

Example: Buchnera aphidicola

B. aphidicola is an endosymbiont of aphids. These animals are sap-sucking parasites of plants, and so their diet is rich in minerals and plant sugars, but essentially devoid of essential vitamins and amino acids. They have a unique organ, the bacteriome, consisting of about 70 bacteriocytes. These are specialized cells containing vacuoles filled with B. aphidicola. These endosymbiotic bacteria provide the insect with the vitamins and essential amino acids it needs for survival; aphids fed antibiotics stop growing and reproducing, and die prematurely. The endosymbionts are transmitted to offspring in utero, and like Wolbachia (see the above discussion of α-proteobacteria) has had a dramatic impact on the reproductive biology of the insect. Aphids are parthenogenic, producing live pregnant offspring without the need for fertilization.

Thiotrichs

These are gliding filamentous sulfur-oxidizers, commonly found in marine and freshwater sediments, and cold sulfur springs. Sulfide is oxidized first to elemental sulfur, which is stored in intracellular granules. When sulfide is depleted, these sulfur granules are oxidized to sulfate. These organisms are microaerophilic, facultative autotrophs, and most are capable of nitrogen fixation. Few have been grown in pure culture, and species identification is based on morphology.

Example: Beggiatoa alba

Beggiatoa alba is found in on the surface of freshwater and marine sediments, but is most conspicuous in freshwater sulfide-rich springs, where it can form spectacular white fuzzy mats covering all submerged surfaces. B. alba is the only formally recognized species of this genus, but a wide range of species have been defined informally. The primary distinction between species is filament diameter; B. alba is 2-3μm is diameter. Unlike other filamentous colorless sulfur Bacteria, no holdfasts or sheaths are present.

Pseudomonads

These organisms are very common in most aerobic environments. They are obligately aerobic except for a few that can grow anaerobically by nitrate reduction, and are straight or slightly curved (not helical) rods with polar flagella. "Pseudo-monas", or "false unit", is an apt name - until recently, this was a huge group with species that turned out to be unrelated gamma and beta proteobacteria. The genus has recently been divided up phylogenetically, and only organisms related to the “fluorescent” Pseudomonas species remain in this genus. “Fluorescent” refers to diffusible pigments produced by these organisms that are siderophores; they bind iron with very high affinity and are used by the organism to scavenge trace quantities of this essential mineral. These organisms are common in lab distilled water, because they are experts at extracting trace amounts of nutrients from sparse environments. Some are opportunistic pathogens (P. aeruginosa is usually the proximal cause of death for cystic fibrosis and burn patients), but most are free-living oligotrophic aquatic species. This group also contains the free-living nitrogen-fixers, the Azotobacteria.

Example: Azotobacter vinelandii

The azotobacteria are free-living nitrogen fixers, distinguished from most species of the genus Pseudomonas only by the ability to fix nitrogen, and distinguished from the rhizobia by the fact that they do not infect plants (although some are external symbionts of roots). Like other members of the genus Azotobacter, A. vinelandii differentiates into resting spore-like microcysts in stationary phase. A. vinelandii is a common soil, freshwater and marine inhabitant, preferring slightly alkaline (pH 7.5-8.0) conditions. Unlike other motile pseudomonads, A. vinelandii has peritrichous flagella.

Purple sulfur Bacteria

The purple sulfur Bacteria (Chromatia) are all phylogenetically and metabolically much alike. They are anaerobic photosynthetizers that require sulfide for growth, and so in some ways resemble the green sulfur Bacteria (Chlorobi), and are often found in the same environment (see Chapter 9 for a discussion of photosynthesis and the Chlorobi). In these environments, the purple sulfur bacteria are often found overlying the green sulfur bacteria because they require more light and less sulfide. Photosynthesis is by cyclic photophosphorylation, and reducing power (NADH) for autotrophic carbon fixation is generated by reverse electron flow using sulfide as the electron donor. Elemental sulfur or polysulfides generated by sulfide oxidation is stored in intracellular globules; when environmental sulfide is depleted, these globules are oxidized first to sulfite and then sulfate. Most purple sulfur Bacteria are also capable of heterotrophic growth in the absence of light. These organisms appear in Winogradsky columns as pastel purple blotches in the sulfide-rich anaerobic regions of the column.

Example: Chromatium vinosum

Chromatium vinosum is a large (ca. 2 x 3-6μm) rod-shaped specie that accumulates many small sulfur globules per cell. Motile by polar flagella, and cells grow individually, not in clumps as do most other purple Bacteria of other genera. Species in this genus are usually distinguished by cell size and absorption spectra. Unlike most other purple sulfur Bacteria, C. vinosum can use hydrogen in place of sulfide as an electron donor for reverse electron flow.

Class δ-proteobacteria

• Class δ-proteobacteria
  • Order Bdellovibrionales
    • Family Bdellovibrionaceae (e.g. Bdellovibrio, Vampirovibrio)
    • Family Bacteriovoracaceae (e.g. Bacteriovorax, Peredibacter)
  • Order Desulfarcales
    • Family Desulfarculaceae (Desulfarculus)
  • Order Desulfovibrionales
    • Family Desulfovibrionaceae (e.g. Desulfovibrio)
    • Family Desulfomicrobiaceae (e.g. Desulfomicrobium)
    • Family Desulfohalobiaceae (e.g. Desulfohalobium, Desulfomonas)
    • Family Desulfonatronumaceae (Desulfonatronum)
  • Order Desulfobacterales
    • Family Desulfobacteraceae (e.g. Desulfobacter, Desulfonema)
    • Family Desulfobulbaceae (e.g. Desulfobulbus, Desulfocapsa)
    • Family Nitrospinaceae (Nitrospina)
  • Order Desulfurellales
    • Family Desulfurellaceae (Desulfurella, Hippea)
  • Order Desulfuromonales
    • Family Desulfuromonaceae (e.g. Desulforomonas, Pelobacter)
    • Family Geobacteraceae (e.g. Geobacter, Trichlorobacter)
  • Order Myxococcales
    • Family Cystobacterineae (e.g. Cystobacter, Stigmatella)
    • Family Myxococcaceae (e.g. Angiococcus, Myxococcus)
    • Family Polyangiaceae (e.g. Chondromyces, Polyangium)
    • Family Nannocystineae (e.g. Nannocyctis)
    • Family Haliangiaceae (Haliangium)
    • Family Kofleriaceae (Kofleria)
  • Order Syntrophobacterales
    • Family Syntrophobacteraceae (e.g. Desulforhabdus, Syntrophobacter)
    • Family Syntrophaceae (e.g. Desulfomonile, Syntrophus)
      

About this Class

Diversity
Most of the δ-proteobacteria are sulfate reducers, and this is probably the primitive phenotype of the group. This being also true of the ε-proteobacteria suggests that these two groups share a common ancestral sulfate-reducing phenotype, either because they are specifically related (share a common branch off of the remainder of the proteobacterial tree) or because sulfate reduction is the primitive phenotype of the proteobacteria in general.

Metabolism
Although a much smaller group than the α-, β- or γ-proteobacteria, like these other groups the metabolisms found in the δ-proteobacteria is very broad. Characterized members of this group fall into three general classes of metabolism; anaerobic sulfate reducers and syntrophic hydrogen-generating heterotrophs, aerobic heterotrophs, and parasites of other Bacteria.

Morphology
Most of the organisms is this group are straight or slightly curved rods.

Habitat
The habitats of these organisms varies with their phenotypes. The sulfate reducers are the most common, and are sometimes predominant members of sulfate-containing anaerobic environments.

Sulfate reducers & hydrogenic syntrophs

Sulfate-reducers are common in many anaerobic environments, but are predominant in marine and estuary sediments (saltwater is rich in sulfate). Metabolism is anaerobic respiration, using organics (or sometimes H2) as the electron donor for electron transport. Sulfate is the terminal electron acceptor. In the environment, this sulfide often reacts with metal cations (chiefly Fe+++) to produce insoluble black metal sulfides. This is the black color typical of marine, estuary, and nutrient-rich freshwater sediments and muds.
Metabolism by the hydrogenic syntrophs is related to that of the sulfate reducers, except that protons are used as the terminal electron acceptor in place of sulfate, to generate molecular hydrogen. This process is energetically favorable only if the ambient concentration of hydrogen is vanishingly low, and so they must live in symbiosis with hydrogen-utilizing organisms, such as methanogens, that consume the hydrogen as quickly as it is generated.

Example: Desulfovibrio desulfuricans

D. desulfuricans are slightly curved rods, motile by a single polar flagellum. Oxidizes hydrogen and a wide range of organics, including glycerol (which most anaerobes cannot utilize). Organics are incompletely oxidized, generating acetate as the primary waste product. When oxidizing hydrogen, they require acetate as a source of carbon for growth; they are not autotrophic. D. desulfuricans can reduce sulfate or sulfite, or even protons; hydrogenic growth requires removal of this hydrogen product, either by thorough flushing of the media or co-cultivation with methanogens (i.e. syntrophically).

Myxobacteria

The myxobacteria are unicellular aerobic gliders (twitching motility is also used) with a complex life cycle, usually found in terrestrial organic-rich environments, especially on bark or decomposing leaves and wood. They grow individually in thin swarming sheets, excreting lytic and digestive enzymes that lyse other Bacteria, on which the myxobacteria feed. When starved, myxobacteria aggregate and develop into fruiting bodies, with base, stalk and spore cells. This is a terminal differentiation; only the spore cells have a future, and so the fruiting body is a true multicellular organism. Spores are released into the environment, & those that blow or wash to a better environment germinate to produce a new crop of free-living cells.

Example: Myxococcus xanthus

M. xanthus is the best-studied member of this group, being the easiest to grow to high density in liquid cultures and being genetically manipulatable. As a result, aggregation and sporangium formation in M. xanthus are model systems for bacterial cell-cell communication, self-organization, and development. It produces simple spheroid fruiting bodies on short stalks.

Parasites of other Bacteria

Although many bacteria are predatory, that is they can kill others and feed on the nutrients released, the Bdellovibrios actually invade or attach to the surface of other bacterial cells and parasitize them. As a result, they grow as plaques on lawns of host Bacteria, much like the plaques produced by viruses. Members of the genus Bdellovibrio can parasitize a wide range of Gram-negative Bacteria (and a few Gram-positives) and take up residence in the periplasmic space of the host. Others, e.g. Vampirococcus, are very host-specific (infecting only Chromatium, in this example) and are epibiotic, attaching to the surface but not entering into the host.

Example: Bdellovibrio bacteriovorans

B. bacteriovorans is probably the best-studied member of this group. Commonly found in soil and freshwater environments, the swarmer “attack-phase” of this organism is a small (0.25-0.4 x 1-2μm) vibrio, motile by a single polar flagellum. This flagellum is unusual in being ensheathed by the outer membrane. After attachment of the attack-phase parasite to a host cell, the parasite looses the flagellum, then passes through the outer membrane of the host and resides in the periplasmic space. In some cases, the parasite can reside quiescently in the host, but more often immediately begin to extract nutrients from the host cell for growth. The parasitic cell grows by elongation; the length of the resulting spiral-shaped parasite depends primarily on the initial size of the host. When the host is spent, the parasite divides into a number of attack-phase cells, which are released by lysis of the host.

Class ε-proteobacteria

• Class ε-proteobacteria
  • Order Campylobacteriales
    • Family Campylobacteraceae (e.g. Campylobacter, Sulfuospirillum)
    • Family Helicobacteraceae (e.g. Helicobacter, Sulfuricurvum, Wolinella)
    • Family Hydrogenomonaceae (Hydrogenomonas)
  • Order Nautiales
    • Family Nautiliaceae (e.g. Caminibacter, Nautilia, Thioreductor)

About this Class

Diversity
Familiar ε-proteobacteria form a relatively narrow phylogenetic group of intestinal symbionts, but the phylogenetic range of environmental members of this group, represented by rRNA sequences extracted primarily from deep-sea environments, suggests that this is in actuality a very large and phylogenetically diverse Class.

Metabolism
These organisms are microaerophilic or anaerobic heterotrophs or chemoautotrophs. Metabolism is by anaerobic respiration using a wide range of organic and inorganic electron donors and acceptors, but generally not carbohydrates. A common feature of the ε-proteobacteria is the ability to use hydrogen as an electron donor for electron transport. Sulfate reduction seems also to be common.

Morphology
Most of the cultivated ε-proteobacteria are helically curved rods, and are motile by polar flagella. The deep-sea environmental ε-proteobacteria have a wide range of morphologies, including vibrio and helical forms, but also rods, cocci, and filaments of all types.

Habitat
The most well-known ε-proteobacteria are intestinal symbionts, but in deep sea environments, and particularly marine hydrothermal zones, the ε-proteobacteria are predominant. Deep sea hydrothermal ε-proteobacteria are abundant not just in water and sediment, but as external and endosymbiotic inhabitants of vent-associated animals.

Intestinal symbionts

Members of the genera Campylobacter, Helicobacter and Wolinella are inhabitants of the upper GI epithelium of mammals and birds. Most are commensalistic, at least in their natural host, but some are pathogens and many cause zoonotic disease. For example, Campylobacter is a common commensal in birds, but in humans is perhaps the single most common cause of foodborne disease.

Example: Helicobacter pylori

H. pylori is a microaerophilic curved rod with several unipolar flagella that are sheathed, with a distinctive bulb at the distal ends. It is a common symbiont of the stomach and duodenal lining, colonizing about 70% of humans. In most cases no symptoms occur and the symbiosis persists for life. In some cases, however, colonization by H. pylori results in gastritis or peptic ulcers, and is a contributing factor for stomach cancer. However, there is evidence that H. pylori may also help modulate stomach acidity and reduce acid reflux.

Deep sea hydrothermal vent-associated species

Although few have been cultivated, molecular surveys of environmental samples show that ε-proteobacteria are very common, even predominant, in many marine hydrothermal vent environments. These environments usually bring to mind the hyperthermophilic Archaea, but the cold area surrounding the hot vents are oases of life both macroscopically and microscopically. In fact, it is the mixture of hot, reduced geothermal water and cold oxygenated sea water, each by themselves more-or-less at equilibrium, that creates the chemical disequilibria that provide the chemical potential energy that can be harvested by lithotrophic organisms. Except in the hottest regions of these vents zones, molecular phylogenetic analysis suggests, ε-proteobacteria are very abundant. This includes the sediments, surfaces, and waters of these regions, but also the symbionts of the animals that inhabit these regions.

Example: The endosymbiont of the scaly snail Crysomallon squamiferum

The scaly snail (Crysomallon squamiferum) is a unique animal found only in Indian Ocean hydrothermal vent fields. Instead of an operculum (the other half of the shell than other snails usually use to cover themselves when retracted), the body of the scaly snail is covered in tough, iron sulfide reinforced scale-like plates. More amazing, the scaly snail has only a vestigial digestive tract and radulus (a scrapping tongue). Instead, the scaly snail has very enlarged esophogeal glands filled with ε-proteobacterial endosymbionts. The animal probably absorbs sulfides from the environment through the crawling surface of its foot; these sulfides are brought to the esophogeal glands along with oxygen absorbed in the gills, Here the sulfide-oxidizing ε-proteobacterial endosymbionts use these to generate energy and fix carbon from CO2. In return for a place to live and a supply of resources, the Bacteria provide the snail with some form of nutrition. The snail, then, is a chemoautotrophic animal. This symbiosis is analogous to that of the giant vent tubeworm (Riftia) and several other hydrothermal vent animals. The symbiotic biofilm covering the snail is predominated by a wide range of ε-proteobacteria, and these presumably participate in the iron sulfide mineralization of the hosts scales and shell surface.

The concept of “Proteobacteria”

A review the basics of electron transport.

Electron transport is carried out by a series of electron carriers in an 'electron transport chain' in the cell membrane. This allows the oxidation half-reaction (electron donation) and the reduction half-reaction (electron accepting) to be physically separated, so that the energy released by the reaction can be captured.

Some electron carriers in the electron transport chain really do carry just electrons, others carry hydrogens (electrons + protons). When an electron carrier transfers an electron to a hydrogen carrier, the hydrogen carrier must capture a proton from solution; when it then transfers the hydrogen to an electron carrier, it releases the proton to solution.

The carriers in the electron transport chain are physically organized in such a way that when a carrier needs to capture a proton (when an electron carrier is donating to a hydrogen acceptor), it gets it from the cytoplasmic side of the membrane. When a carrier needs to release a proton (when a hydrogen carrier is donating to an electron acceptor), it does so at the outside surface of the membrane. The result, then, of electron transport down the chain is that protons are collected from inside and released to the outside of the membrane. In this way, the chemical energy of the oxidation/reduction reaction mediated by the chain is captured in the form of a proton gradient, rather than being entirely lost to heat as would occur if the reaction occurred in solution.

The energy in this proton gradient is in two forms: a chemical gradient (high protons outside, low protons inside) and an electrical potential (positive outside, negative inside). The energy in the proton gradient is converted back to chemical energy, which the cell can use, by ATPase. ATPase (named for the reverse reaction) is a membrane protein that leaks the protons back into the cell, using the energy of the proton gradient to phosphorylate ADP to make ATP.

The Proteobacteria

The 'purple bacteria & relatives' really are 'Proteobacteria'; they seem to have to change readily (in evolutionary terms) between sulfur oxidation or reduction, photosynthesis, heterotrophy, nitrogen oxidation or reduction, &c, &c. All of these lifestyles are based on the same electron transport chain, all that's changed are the inputs and outputs, i.e. the electron donors and acceptors of the oxidation/reduction reaction from which they derive energy.

Most heterotrophs oxidize organic compounds into CO2 to generate NADH, which serves as the electron donor for electron transport (and ultimately ATP synthesis), using O2 as the terminal electron acceptor. Sulfur oxidizing autotrophs use H2S as an electron donor (converting it to sulfate, if completely oxidized) and oxygen as the electron acceptor (generating water). Other electron donors are thiosulfate, elemental sulfur, activated photosystem chlorophylls, hydrogen, methane, and ammonia. Other electron acceptors are sulfate, sulfite, sulfur, nitrite, nitrate, ferric ion (and many other oxidized metal ions), and reduced photosystem chlorophylls. For the production of a proton gradient for ATP synthesis, the electron flow is in the 'forward' direction. Reverse electron flow is generally reserved for the synthesis of NADH by autotrophs (who don't use organic carbon to make NADH) for reducing CO2 to organic carbon (see Chapter 9).

Because all of these metabolisms are base on the same electron transport change, all that’s needed to change metabolic phenotype is to change either the enzyme that catalyses the oxidation reaction (feeding electrons into the beginning of electron transport chain) or the reduction reaction (transferring electrons from the end of the electron transport chain to the terminal electron acceptor) or both. For example, the acquisition of a single polypetide, nitrate reductase, could convert a heterotroph from obligate aerobe into an anaerobic nitrate reducer.

The proteobacteria seem to be particular good at changing the inputs and outputs of the electron transport chain, thus the latin ‘changeable Bacteria’. The genes for these enzymes feeding into and out of the electron transport chain are presumably most frequently acquired by horizontal transfer. The proteobacteria may be particularly good at acquiring such useful genes from other sources, and perhaps they are more readily able to accommodate such foreign enzymes into electron transport chain function.

In any case, the metabolic phenotype of an organism can clearly be a superficial trait, and is certainly not a reliable guide to phylogenetic relationships.

Questions for thought

• Make as long a list of Gram-negative Bacteria as you can from your memory or old notes. Now look them up; what fraction of them are proteobacteria? What fraction are α-, β-, γ-, δ- or ε-proteobacteria?
  
  
• What complex bacterial life cycles are you familiar with other than the ones described in this lecture?
  
  
• The cells of Caulobacter have a specified polarity; each end is distinct. Swarmer cells have a flagellum at only one end, and the receptors for chemotaxis are also localized to this end. Stalked cells have a stalk at only one end, and daughter cells are produced by fission from the other end. What other Bacteria are you familiar with that have defined polarity? Which ones don’t? Are you sure? If Bacteria have terminal (end-to-end) polarity, what about dorsal/ventral and lateral polarity?
  
  
• What do you think you'd see if you made a time-lapse close-up video of growing films of Myxococcus? What about growing colonies of E. coli? Bacillus? Are you sure?
  
  
• Do you know of any examples where the entire ecological niche of an organism is defined by one or a few genes it acquired horizontally? How might you recognize these cases?
  
  
• Imagine a segment of DNA was recently acquired by an organism and integrated into its genome. One of the genes in this segment conferred a useful trait on its new host, the rest did not. How might a scientist studying this organism recognize that this is a foreign gene? How might this gene change in the time shortly after being acquired? What might happen to the other, non-helpful acquired genes?