Vaccari D.A., Strom P.F., Alleman J.E. Environmental Biology for Engineers and Scientists

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DNA can be speciﬁed either by any single nucleotide or by the sum of either A þ TorGþ

C. By convention, % G þ C is used as an indication of genetic relatedness. Organisms

with very different % G þ C compositions cannot be closely related. On the other

hand, % G þ C does not indicate the speciﬁc sequences of the bases, so that organisms

with similar % G þ C values are not necessarily related.

Figure 10.17 Fatty acid methyl ester (FAME) proﬁles showing different patterns for (a) Serratia

marcescens and (b) Tsukamurella paurometabolum. (Courtesy of Michael Fleming.)

238

MICROBIAL GROUPS

DNA Hybridization One means of genotyping cells is by comparing the similarity of

sequences of bases in their DNA. To compare two organisms, the DNA of one is ﬁrst

labeled (often with radioactive

P). The DNA extracted from both organisms is then bro-

ken mechanically into small fragments and heated to separat e the two complemen tary

strands (denatured). The labeled DNA and an excess of the unlabeled DNA are next

mixed together and cooled to allow them to reanneal (re-form a double strand). The

extent to which the two different DNAs are able to combine with each other, or hybridize,

is a measure of how similar the base sequences are. This can be determined by the amount

of radioactivity present in the reannealed DNA.

Molecular Probes A large number of molecular probing techniques are now available or

under development. One group of methods of particular interest is referred to as ﬂuores-

cent in situ hybridization (FISH). In a generalized FISH approach, an oligonucleotide

(short chain of nucleotides, typically 15 to 25 bases for FISH) containing a sequence char-

acteristic of a particular taxon of interest is labeled with a ﬂuorescent dye. The labeled

oligonucleotide is then allowed to hybridize with the complementary sequence in the

organism in situ. With ﬂuorescence microscopy, it is then possible to see the numbers

and location of the brightly colored target organism within a natural community. Depend-

ing on the degree of speciﬁcity of the sequence chosen, this approach potentially can be

used to identify organisms belonging to any taxonomic level ranging from domain (e.g.,

Bacteria) to a particular species or even strain.

Another type of probe is used in ribotyping. Restriction enzymes break DNA at points

where speciﬁc sequences of nucleotides occur. If DNA is treated with one or more such

enzymes, fragments of DNA are produced. These fragments are then separated by size on

a gel and probed with 16S rRNA genes. For treatme nt with a particular set of restriction

enzymes, the resulting pattern of DNA fragment sizes will be characteristic of the organ-

ism.

Denaturing gradient gel electrophoresis (DGGE) analysis offers a characterization of

the GC composition of DNA segments coding for rRNA or other genes. Following tagging

and ampliﬁcation using PCR (Section 6.3.3), these fragments are drawn electrophoreti-

cally (i.e., attracted by their electrical charge) through gel tracks laden with a gradient

matrix of a denaturing agent (e.g., urea ranging from 30 to 55%). Since the G–C triple

bond is stronger than the A–T double bond, it does not denature until farther along in the

gradient. Fully denatured fragments stop migrating, resulting in a linear separation of the

fragments into a banding pattern that is characteristic of the organism (Figure 10.18). A

similar approach is used in thermal gradient gel electrophoresis (TGGE), except that a

temperature gradient is used for denaturing the DNA.

16S rRNA Analysis The primary tool used today in determining phylogeny is 16S

rRNA analysis, developed largely by Carl Woese beginning in the early 1970s. Prokaryo-

tic ribosomal RNA (rRNA) is composed of three molecules, referred to by their sedimen-

tation coefﬁcients measured in Svedberg units (S) as 5S (containing 120 bases), 16S

(1500), and 23S (2900). Woese initially worked with 5S fragments but found they

were too small to yield sufﬁcient information. The emphasis then shifted to 16S rRNA

(and the comparable 18S rRNA found in eukaryotes) based on its larger, yet still manage-

able number of nucleotides.

One important advantage of using the nucleotide sequences of 16S (or 18S) rRNA for

determining relationships is that these molecules occur in all organisms. Also, because of

MICROBIAL TAXONOMY 239

their central role in the process of gene expression, there are strong selective pressures,

making it unlikely that they will undergo rapid changes. This leads to the presence of

extended regions of the molecule that are highly conserved (little changed) and which

therefore are useful for establishing distant phylogenetic relationships. On the other

hand, they also have an adequate number of variable regions that can be used to examine

closer relationships. Thus, 16S rRNA can be used as an evolutionary clock, or chron-

ometer, to measure the phylogenetic distance between taxa at a variety of levels based

on the number of nucleotide differences—representing stable mutations—that have

occurred over time.

One method to sequence 16S rRNA begins with extraction of a cell’s RNA. A small

DNA oligonucleotide primer (15 to 20 nucleotides in length) that is complementary in its

base sequence to a conserved region of the 16S rRNA molecule is added. Reverse tran-

scriptase can then be used to generate complemen tary DNA (cDNA), which in turn is

ampliﬁed using PCR. The nucleotide sequences of these cDNA are then determined,

and the original sequence of the rRNA is deduced from them. Another common approach

is to extract and sequence the DNA of the gene that codes for the 16S rRNA rather than

the RNA itself.

The 16S rR NA sequences of thousands of species have now been determin ed. Using

advanced computer algorithms, short (6 to 10 nucleotides) signature sequences have

been found that are highly consistent within groups of organisms. Also, phylogenetic

trees can be generated in which the evolutionary distance between two groups is indicated

by the length of the connecting lines (Figure 10.19).

Figure 10.18 Denaturing gradient gel extraction track proﬁles.

240

MICROBIAL GROUPS

10.5 BACTERIA

As a group, the domain Bacteria is extremely diverse, including phototrophs and chemo-

trophs, organotrophs and lithotrophs, heterotrophs and autotrophs, aerobes and anaerobes,

psychrophiles and mesophiles and therm ophiles and hyperthermophiles, halophiles and

nonhalophiles, acidophiles and neutrophiles and alkaliphiles, saprophytes and parasites.

They are able to utilize a vast array of organic compounds (i.e., all naturally occurring

and almost all synthetic organics) as carbon and energy sources, many reduced inorganics

as electron donors (for energy), and many oxidized inorganics as electron acceptors.

One way of discussing microorganisms is by function, grouping together all of those

with a particular ability or characteristic (e.g., phototrophs). However, sometimes organ-

isms that have similar functions are actually quite different from each other. (This is

origin

pJP 27

pJP 78

Marine

group I

Pyrodictium

Thermoproteus

Sulfolobus

Thermofilum

Methanopyrus

Thermococcus

Methanococcus

Methanobacterium

Methanosarcino

Methanospirillum

Holoferax

Chramatium

E. coli

Riftia symbiont

Agrobacterium

Mitochondria

Chlorobium

Cytophaga

Epulopiscium

Bacillus

Synechococcu

Thermus

Thermomicrobium

Thermologa

Aquifex

Chloroplast

EM 17

0.1 changes per nuclcotide (nt)

EUKARYA

croscopic

organism

Zea

Homo

Coprinus

Paramecium

Porphyra

Dictyostelium

Entamoeba

Naegleria

Euglena

panosoma

Physarum

Encephalitozoon

Vairimorpha

Hexamita

Giardia

Tritrichomonas

ARCHAEA

BACTERIA

Figure 10.19 Phylogenetic tree indicating evolutionary branching and distance between groups

based on on rRNA analysis. Fungi are represented by Coprinus (a mushroom), plants by Zea (corn),

BACTE RIA 241

referred to as convergent evolution, since they have approached a similar form from dif-

ferent starting points.) Thus, microbiologists generally prefer, where possible, to group

bacteria based on phylogeny. That is the approach that is taken here, for the most part,

although it should be recognized that with our growing knowledge, these groupings con-

tinue to change.

Figure 10.19 showed the phylogeny of Bacteria using an approach based mainly on

16S rRNA analysis. The domain was broken into about eight known major groupings

(although other 16S rRNA sequences have been found in environmental samples, indicat-

ing that additional ‘‘unknown’’ groups also exist). What should these major groupings of

Bacteria (and Archaea) be called?

Previously, they may have been referred to as phyla, or divisions (reﬂecting bacterio-

logy’s roots in botany), or commonly, groups (reﬂecting phenotypic similarities rather

than phylogenetic relationships). However, in looking at phylogenetic trees, such as

that in Figure 10.19, som e microbiologists have come to believe that if the differences

between plants and animals (and fungi) warrant assignment to groupings at the level of

kingdoms, the much greater differences among bacteria should also.

Of course, not all microbiologists agree, and this has not yet been widely accepted

among other biologists, who traditionally have focused on Eukarya, and particularly,

plants and animals. One argument is that 16S (or 18S for eukaryotes) rRNA analysis is

not necessarily the ‘‘one true’’ measure of phylogeny and degree of evolutionary diver-

gence. Still, as more is learned about microbial diversity using a variety of tools, it seems

quite possible that some cat egorization scheme like this eventually will be accepted. Thus

for Chapters 10 through 13, where microbiology is emphasized, these groupings will be

referred to as ‘‘Kingdoms,’’ even though elsewhere we do not make this distinction.

Table 10.3 shows 14 major groupings of Bacteria and indicates some further subdivi-

sions into classes and orders. The newest (second) edition of Bergey’s Manual of Systema-

tic Bacteriology decided not to refer to the major groups as kingdoms, and instead, listed

23 phyla. (The term domain for Bacteria was retained; there had been a proposal to use

‘‘empire’’ instead.) The table also includes a number of representative, interesting, and

environmentally important genera. Comparison with the phylogenetic tree of

Figure 10.19 shows many similarities, but also some differences reﬂecting the changing

taxonomy of bacteria.

Some of the kingdoms have few genera, perhaps none of which are of known major

importance in environmental engineering and science. Other kingdoms have numerous

genera of great relevance to humans. The three largest are Proteobacteria, Firmicutes

(gram positives), and Cyanobacteria. It should also be noted that a number of bacteria

(generally not shown in the table) do not appear to ﬁt in any of the kingdoms and are

thus of ‘‘uncertain’’ afﬁliation. (One extreme example is the genus Chrysiogenes, contain-

ing only one recognized species, which may merit its own kingdom!) Remember, these

divisions are based on similarities in the genetic code, not on any attempt to distribute

species equally among groups. One way to look at this unevenness (which also occurs

among plants and animals) is to realize that some approache s to survival lend themselves

to greater diversiﬁcation because of the range of niches available.

It should also be recognized that our knowledge of bacterial groups is uneven. Natu-

rally, because more effort has gone into their study, we tend to know more about groups

that appear to be of greater importance to us. This includes bacteria used comme rcially, as

well as pathogens of humans, domestic animals, and crop plants. On the other hand, it is

not unusual to ﬁnd that an organism isolated from the natural environment—or even a

242 MICROBIAL GROUPS

TABLE 10.3 The Bacteria: A Proposed Phylogeny Including

Some Representative, Interesting, and Environmentally

Important Genera

Kingdom 1. Aquaﬁcae

Class 1. Aquaﬁcae

Aquifex

Class 2. Thermotogae

Thermotoga

Class 3. Thermodesulfobacteria

Thermodesulfobacterium

Kingdom 2. Xenobacteria

Class 1. Deinococci

Deinococcus

Class 2. Thermi

Thermus

Nitrospira (or perhaps a separate Kingdom)

Leptospirillum

Magnetobacterium

Kingdom 3. Thermomicrobia

Class 1. Chloroﬂexi—green nonsulfur bacteria

Order 1. Chloroﬂexales

Chloroﬂexus

Order 2. Herpetosiphonales

Herpetosiphon

Class 2. Thermomicrobia

Thermomicrobium

Kingdom 4. Cyanobacteria—blue-green bacteria

(formerly, blue-green algae)

Class 1. Prochlorophyta

Order 1. Chroococcales

Chroococcus

Prochloron

Synechococcus

Order 2. Pleurocapsales

Pleurocapsa

Order 3. Oscillatoriales

Lyngbya

Oscillatoria

Spirulina

Order 4. Nostocales

Anabaena

Calothrix

Nostoc

Order 5. Stigonematales

Stigonema

Kingdom 5. Chlorobia—green sulfur bacteria

Chlorobium

Kingdom 6. Proteobacteria

Class 1. a-Proteobacteria (Rhodospirilli)

Class 2. b-Proteobacteria (Neisseriae)

(Continued )

BACTE RIA 243

sewage treatment plant—is not a previously known, or at least not well described, species.

Also, our knowledge of bacteria has been limited by our ability to culture (grow) them in

the laboratory; those that are not readily grown on laboratory media have been difﬁcult to

study. Interestingly, we do know a fair amount about thermophilic bacteria. This is at least

in part because of the early efforts of Thomas D. Brock, who began studying hot springs

with the idea that the extreme conditions limited the microbia l diversity, making microbial

ecology studies simpler!

TABLE 10.3 (Continued )

Class 3. g-Proteobacteria (Zymobacteria)

Class 4. d-Proteobacteria (Predibacteria)

Class 5. e-Proteobacteria (Campylobacteres)

Kingdom 7. Firmicutes—Gram positives

Class 1. Clostridia

Class 2. Mollicutes

Class 3. Bacilli

Class 4. Actinobacteria

Kingdom 8. Planctomycetacia

Class 1. Planctomycetacia

Order 1. Planctomycetales

Planctomyces

Order 2. Chlamydiales

Chlamydia

Kingdom 9. Spirochetes

Borrelia

Leptospira

Spirochaeta

Treponema

Kingdom 10. Fibrobacteres

Fibrobacter

Kingdom 11. Bacteroidetes

Bacteroides

Kingdom 12. Flavobacteria

Flavobacterium

Kingdom 13. Sphingobacteria

Cytophaga

Flexibacter

Haliscomenobacter

Saprospira

Kingdom 14. Fusobacteria

Fusobacterium

Streptobacillus

Kingdom 15. Verrucomicrobia

Prosthecobacter

All classes listed if more than one. All orders listed if more than

one except for the two phyla Proteobacteria and Firmicutes. Families not

shown. (Based in part on outline available from Bergey’s Manual Trust.)

Large important group; additional breakdown and genera given in Tables

10.5 (Proteobacteria) and 10.7 (Firmicutes).

244 MICROBIAL GROUPS

Phylogenetic grouping, as in Table 10.3 (and Figure 10.19), does not always lend itself

to neat phenotypic groupings of organisms with similar characteristics. Still, we mainly

use this organization below for a brief description of the Bacteria. No attempt is made to

cover every subgroup or genus, and many details are omitted.

10.5.1 Aquaﬁcae

This kingdom, which is considered to be deeply rooted (close to the original bacterial

forms), is so diverse that there are proposals to break it into three (or alternatively, to

lump it with the next). The known species are thermophilic or hyperthermophilic. Aquifex

is the most thermophilic known true bacteria (optimum 85



C, maximum 95



C) and is a

chemolithotrophic autotroph (growing on H

,orS

2

). It requires oxygen or nitrate

as an elect ron acceptor but cannot tolerate high O

concentrations. Thermotoga is also a

hyperthermophile but is a fermentative chemoorganotroph. Thermodesulfobact erium is a

thermophilic sulfate reducer using fermentation products such as lactate and pyruvate as

its carbon and energy source.

Although they are undoubtedly of signiﬁcance in the environments in which they are

found (mainly hot springs and marine thermal vents), these organisms are generally not of

concern to environmental engineers and scientists. However, their unique features make

them of special interest to microbiologists studying the evolution of life or biochemical

diversity.

10.5.2 Xenobacteria

This kingdom contains two classes, Deinoc occi and Thermi. Deinococcus radiodurans

was ﬁrst isolated from food that had been irradiated to sterilize it. It is extraordinarily

radiation resistant, able to withstand more than 1.5 million rad of gamma irradiation

(3000 times the lethal dose to humans). Many deinococci, which are frequently reddish

in color due to caretenoid pigments, also are resistant to ultraviolet radiation and other

mutagens and are able to tolerate drying. Their resistance to genetic damage appears to

result in large part from an exceptional ability to repair damaged DNA rapidly. It is hoped

that these bacteria may prove useful in the future for certain types of hazardous waste site

cleanups.

Thermus aquaticus, ﬁrst found in hot springs in Yellowstone National Park by a group

led by Thomas Brock, was one of the ﬁrst extreme thermophilic organisms isolated. It has

also been found in hot-water heaters. Its heat-stable Taq DNA polymer ase is the enzyme

commonly used in PCR (Section 6.3.3).

Nitrospira is an autotroph that derives energy from the oxidation of nitrite to nitrate.

Thus, it is often grouped with the other nitrifying bacteria (Proteobacteria; see

Section 10.5.6). However, it also has been proposed to include it in the class Thermi,

or perhaps in its own kingdom, based on phylogeny.

10.5.3 Thermomicrobia (Including Green Nonsulfur Bacteria)

Although Thermomicrobium is a thermophilic (75



C optimum) chemoorganotroph, most

of the known members of this small kingdom are phototrophic green nonsulfur bacteria.

Chloroﬂexus is an anoxygenic (non-oxygen producing) phototroph that can grow autotro-

phically but grows better as a photoheterotroph (using organic carbon sources). It also can

BACTE RIA 245

grow aerobically as a chemoorganotroph. It is thought to be descended from the earliest

phototrophs. Table 10.4 compares several characteristics of the various groups of photo-

trophic bacteria.

10.5.4 Cyanobacteria: Blue-Green Bacteria (Formerly, Blue-Green Algae)

The Cyanobacteria is a large, diverse, and environmentally important bacterial group. As

discussed in Section 10.1, their early forms probably were responsible for producing the

oxygen of Earth’s atmosphere, allowing the development of advanced eukaryotic life

forms. Their present relevance to environmental engineers and scientists includes their

role in the eutrophication of lakes and the production of tastes and odors in drinking

waters drawn from surface supplies.

Because they are oxygen-producing photoautotrophs, cyanobacteria were long consid-

ered to be algae (Cyanophyta). However, once it was realized that they were prokaryotes,

they were renamed and reclassiﬁed. It is now believed that they probably shared a com-

mon ancestor with chloroplasts. They are compared with the other phototrophic bacteria

in Table 10.4. Their green color comes from chlorophyll a, while the blue color comes

from other pigments called phycocyanins.

Most Cyanobacteria are strictly photoautotrophic, but a few can utilize simple organics

as carbon sources (photoheterotrophs) or even grow in the dark as chemoorganotrophs.

Many (e.g., Oscillatoria) have gliding motility. Although they are usually thought of as

aquatic freshwater organisms, they are also found in the ocean and in soils. A number can

withstand extreme environments, such as hot springs with temperatures of 70



C, the sur-

face of deser t soils, bare rock, saline lakes, and shallow tidal seas (where they may form

large mats). Different species grow as individual cells, ﬁlaments, or various types of

aggregates.

Many Cyanobacteria have the unusual ability to be able to ﬁx nitrogen (convert N

to a

combined form, usually as ammonium or an amine compound). Interestingly, nitrogen-

ase, the enzyme responsible for nitrogen ﬁxation, is sensitive to oxygen; this means that

nitrogen ﬁxation cannot readily occur in the light (when oxygen is being produced) in

most species. However, some ﬁlamentous Cyanobacteria (e.g., Anabaena, Figure 10.20)

form differentiated cells, known as heterocysts, for nitrogen ﬁxation. These special struc-

tures do not photosynthesize and have thickened walls to minimize oxygen diffusion from

the outside. They receive nutrition from neighboring cells in the ﬁlament, and in return

make ﬁxed nitrogen available to them.

The ﬁve major groups of Cyanobacteria of Table 10.3 correspond to general morpho-

logical forms: Chroococcales, single cells or small aggregates; Pleurocapsales, small

spherical cells formed through ﬁssion (for repr oduction); Oscillatoriales, ﬁlaments;

Nostocales, ﬁlaments with heterocysts; Stigonematales, branched ﬁlaments. Except for

the Chroococcales, which are very diverse, these groupings also hold up fairly well to

phylogenetic analysis.

10.5.5 Chlorobia: Green Sulfur Bacteria

Another distinct group of phototrophs included in Table 10.4 is the green sulfur bacteria.

Like the green nonsulfur bacteria, they are anoxygenic, and can use hydrogen sulﬁde

S) as their electron donor. The sulﬁde is oxidized ﬁrst to elemental sulfur, which pro-

duces granules outside the cell, and then to sulfate (SO

2

). Also, they contain unique

246 MICROBIAL GROUPS

TABLE 10.4 Characteristics of Phototrophic Bacteria

Green Sulfur Green Nonsulfur Purple Sulfur Purple Nonsulfur Blue-Green Heliobacteria

Kingdom Chlorobia Thermomicrobia g-Proteobacteria a -orb-Proteo-

bacteria

Cyanobacteria Firmicutes

Light Obligately Usually Obligately Usually Aerobic, Obligately

metabolism anaerobic, photo

autotroph (some

also photo

heterotophic)

anaerobic,

photoheterotroph

or photoautotroph

anaerobic,

photoautotroph

anaerobic, photo-

heterotroph or

photoautotroph

photoautotroph anaerobic,

photoheterotroph

Electron

donor for

lithotrophy

S, S H

O 

Dark None Chemoorgano- None Chemoorgano- None Chemoorgano-

metabolism heterotroph heterotroph (some are

chemoorgano-

heterotrophs)

heterotroph

Carbon

source

or organic

carbon

or organic

carbon

(some can use

organic carbon)

Organic carbon

Sulfur Outside cell None Within cell None None None

deposition

Fixation Yes No Yes Yes Yes (many) Yes

Endospores No No No No No Yes

Chlorophyll Bact.-a, and

c, d,ore

Bact.-c Bact.-a, b Bact.-a, b Chlor.-a Bact.-g

247