Molecular Biology Microbial

Harry J. Flint

Rowett Research Institute, Aberdeen, U.K.

INTRODUCTION

The powerful array of techniques that constitute molecular biology arose largely from the study of microorganisms. The ability to construct genomic DNA libraries and cDNA libraries derived from expressed mRNA in bacterial and fungal hosts remains a key approach for isolating genes. Dramatic technical developments, however, have now brought about a further revolution. In particular the polymerase chain reaction (PCR) allows precise amplification of DNA sequences, whereas developments in DNA sequencing, microarray, and proteomics technologies are making it more efficient to deal with whole microbial genomes rather than to search for individual genes. Molecular biology now pervades all areas of microbiology and is producing major advances in our understanding of the diversity and dynamics of the microbial ecosystems found in the animal gut, on plant surfaces, and in soils. Genes are being uncovered that define the interactions between microbes and animal hosts, notably the mechanisms involved in pathogenesis, survival, and the mutualistic relationships that allow herbivorous animals to gain energy from plant material. Molecular information underpins the quest to treat and prevent infectious diseases in animals, track and suppress microbes harbored by animals that cause disease in humans, optimize animal production, and minimize pollution.

MICROBIAL DIVERSITY Ribosomal RNA

It has been difficult for microbiologists to know whether they can culture the full range of microorganisms present in a given habitat, but molecular approaches are now revealing the extent of previously unknown diversity. Most ''culture-independent'' approaches involve the sequencing of ribosomal genes that are amplified directly by PCR from environmental samples. Ribosomal genes (particularly those coding for the small subunit rRNA 16S in prokaryotes, 18S in eukaryotes) are suitable because they occur in all living organisms and contain highly conserved sequences (which are useful for such things as designing ''universal'' eubacterial or archaeal primers for PCR amplification) as well as regions that vary between strains and species. In soils, less than 1% of microbial rRNA sequence diversity appears to be represented by cultured species.[1] Analyses performed on the microbiota of the rumen and the pig and horse large intestine (Fig. 1) reveal enormous diversity; only 17% of eubacterial sequences recovered from the pig, and only 11% from the horse, correspond to known species.[2,3] The rapidly expanding sequence databases allow the design of probes and primers, specific to particular groupings, that are suitable for enumeration by dot blot hybridization, whole-cell fluorescent in situ hybridization (FISH), or real-time PCR.[4] Microarrays are also being developed in which panels of specific oligonucleotide probes can be used to describe the composition of microbial ecosystems.

Molecular profiling approaches such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), again usually based on amplified ribosomal sequences, are widely used to follow shifts in the composition of microbial communities. These produce bands characteristic of different DNA% G+C contents or sequences using primers that target broad phylogenetic groupings. These methods are contributing to our understanding of such topics as the impact of host variation and of diet upon the gut microflora and the impact of management practices upon soil microbial communities.

Gene Tracking

Polymerase chain reaction methods can be used to detect a variety of specific genes in environmental samples without prior cultivation and isolation of microorganisms. They have been applied particularly to virulence determinants (e.g., toxin genes) providing information on pathogen contamination in the food chain. PCR tracking of antibiotic resistance genes in the environment and in human and animal gut bacteria has implications for the debate over the impact of antibiotic use in animal husbandry, which centers on resistance to the antibiotics used in clinical and veterinary medicine.

Eubacterial diversity assessed from PCR amplified 16S ribosomal RNA gene sequences

Rumen

Horse

Fig. 1 Eubacterial diversity in gut samples as determined by amplification and sequencing of 16SrRNA genes. Data are from bovine rumen, horse large intestine, and pig intestine. Clos Eub=Clostridium/Eubacterium/Ruminococcus relatives; Bac Lac=Bacillus/ Lactobacillus/Streptococcus; CFB=Cytophaga/Flavobacterium/Bacteroides. While independent of culture bias, it should be noted that PCR bias and rRNA gene copy number influence the apparent proportions of different types obtained by this approach. (From K. Tajima et al., 1999 cited in Ref. 4 and Refs. 2,3.)

√úCIos-Eub

S Bac-Lac

^CFB

E3 Others

Horse

Fig. 1 Eubacterial diversity in gut samples as determined by amplification and sequencing of 16SrRNA genes. Data are from bovine rumen, horse large intestine, and pig intestine. Clos Eub=Clostridium/Eubacterium/Ruminococcus relatives; Bac Lac=Bacillus/ Lactobacillus/Streptococcus; CFB=Cytophaga/Flavobacterium/Bacteroides. While independent of culture bias, it should be noted that PCR bias and rRNA gene copy number influence the apparent proportions of different types obtained by this approach. (From K. Tajima et al., 1999 cited in Ref. 4 and Refs. 2,3.)

Strain Typing

A plethora of DNA-based methods are now available that enable more precise strain identification among the better known cultivable microorganisms, especially pathogens. Many rapid typing techniques rely on PCR amplification of randomly primed sequences, repeated sequences, or on ribotyping based on ribosomal RNA sequences. Alternatively, the whole genome can be profiled after restriction enzyme cleavage into large fragments that are separated by pulsed field gel electrophoresis (PFGE).

GENOMICS Complete Genomes

Complete microbial genomes range in size from around 1 to 8 Mb for bacteria, and from 10 to 150 Mb for eukaryotes such as yeasts and protozoa. There has been an explosion in genome sequence information that now extends to most human and many animal bacterial pathogens (Table 1).[5] One important outcome has been to identify large genetic regions, or pathogenicity

Table l Some examples of fully sequenced bacterial genomes

Chromosome (plasmid)

Table l Some examples of fully sequenced bacterial genomes

Chromosome (plasmid)

Species, strain

Size base pairs

Predicted protein-coding genes

DNA % (G + C)

Streptomyces coelicolor A3(2)

8,667,507

7,825

71.1

Bacteroides thetaiotaomicron VPI5482

6,260,361 (33,038)b

4,779 (38)

42.8 (47.2)

Escherichia coli K12

4,639,221

4,288

50.8

Clostridium tetani E88a

2,799,250 (74,082)b

2,372 (61)

28.6 (24.5)

Bifidobacterium longum NCC2705

2,256,646

1,730

60.0

Campylobacter jejuni NCTC11168a

1,641,481

1,654

30.6

Mycoplasma pulmonisa

963,879

782

26.6

aPathogenic strain. bPlasmid.

aPathogenic strain. bPlasmid.

islands, that make particular strains infectious. The first projects are now underway to sequence genomes of mutualistic and commensal microorganisms from the animal gut.

Microarrays and Gene Expression

New technologies allow large numbers of sequences, e.g., representing all genes from a given species, to be arrayed on glass slides (microarrays) or on membranes. Genes showing differential expression can then be identified by comparative DNA hybridization. Allied to genome sequencing and rapidly developing proteomic methods for two-dimensional separation and identification of polypeptides, this provides unprecedented power for studies on microbial gene regulation.1-6-1

Metagenomics

Another powerful new approach, metagenomics, involves creating gene libraries from the DNA recovered from a mixed microbial community, followed by screening for specific functions. This allows the recovery of valuable and important genes from microorganisms that may never have been cultivated.1-7-1

GENE TRANSFER AND GENETIC ANALYSIS Natural Gene Transfer Mechanisms

Microorganisms, especially bacteria, frequently carry genetic elements that are capable of transfer between cells independently of the main chromosome. Such elements, which include extrachromosomal plasmids and chromosomally located conjugative transposons, possess genes that promote their own transfer by cell cell contact (conjugation), or that allow them to be mobilized by other elements. In addition many bacteria are able to take up DNA from their environment resulting in natural genetic transformation, or may acquire genes via bacteriophage virus-mediated transduction. Such genetic exchanges play a major role in microbial evolution. Traits including antibiotic resistance, heavy metal resistance, virulence factors, adhesion properties, substrate utilization pathways, and the production of antimicrobials show evidence of natural horizontal transfer.

Genetic Analysis

Mobile genetic elements provide the basis for many molecular biology procedures, in particular, as vectors. In addition, transposons that insert randomly in the genome are used for insertional mutagenesis to identify microbial gene function. In many bacteria and fungi it is relatively straightforward to introduce and over express foreign genes through conjugal transfer or transformation, and to perform targeted gene knockouts. Refinements of these techniques allow the identification of genes that are switched on in particular environments, e.g., in mammalian host tissues (IVET, in vivo expression technology).1-8-1 However, the lack of convenient gene transfer systems remains an obstacle to research in many less-studied species, including most anaerobes. Transformation can be induced artificially in bacteria, e.g., by electroporation, but endogenous nucleases normally present in wild-type strains tend to destroy the incoming DNA.

BIOTECHNOLOGY

Modified (mutant or genetically manipulated) strains of bacteria and fungi are used in a contained manner in the production of enzymes and other products (e.g., amino acids) used as animal feed additives or feed pretreatments. Modified microbial strains have also been considered for other applications that would require their release into the environment, e.g., as silage additives, probiotics, or for bioremediation. These latter possibilities are clearly subject to detailed risk assessments and to public acceptance. Recombinant vaccines and antibody engineering are increasingly important in the treatment and prevention of infectious diseases in animals caused by a wide range of viruses, bacteria, fungi, and protozoa. Again, genomic and proteomic approaches are helping to define new antigens, as well as possible targets for the development of new antimicrobial agents. Microbial genes continue to provide valuable proteins and enzymes for research, industry, and medicine.[9]

CONCLUSIONS

Molecular techniques are revolutionizing our understanding of the diversity and functioning of microbial ecosystems associated with the mammalian gut, soils, and wastes. These techniques provide new tools for tracking and identifying individual genes and species. Complete genome sequences are available for many human and animal pathogens and should soon become available for more nonpathogenic bacteria and eukaryotic microorganisms. The potential benefits for disease prevention, animal production, and environmental management are enormous, although many will take some time to be realized.

ACKNOWLEDGMENT

The author wishes to acknowledge the support of the Scottish Executive Environment and Rural Affairs Department.

ARTICLES OF FURTHER INTEREST

Antibiotics: Microbial Resistance, p. 39 Genetics: Molecular, p. 466 Genomics, p. 469

GI Tract: Animal/Microbial Symbiosis, p. 449 Rumen Microbiology, p. 773

REFERENCES

1. Amman, R.I.; Ludwig, W.; Schleifer, K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 1995, 59 (1), 143 169.

2. Leser, T.D.; Amenuvor, J.Z.; Jensen, T.K.; Lindecrona, R.H.; Boye, M.; Moller, K. Culture independent analysis of gut bacteria: The pig gastrointestinal tract revisited. Appl. Environ. Microbiol. 2002, 68 (2), 673 690.

3. Daly, K.; Stewart, C.S.; Flint, H.J.; Shirazi Beechey, S.P.

Bacterial diversity within the equine large intestine as revealed by molecular analysis of cloned 16S rRNA genes. FEMS Microbiol. Ecol. 2001, 38 (2 3), 141 151.

4. Tajima, K.; Aminov, R.I.; Nagamine, T.; Matsui, H.; Nakamura, M.; Benno, Y. Diet dependent shifts in the bacterial population of the rumen revealed with real time PCR. Appl. Environ. Microbiol. 2001, 67 (6), 2766 2774.

5. Bruggeman, H.; Baumer, S.; Fricke, W.F.; Wiezer, A.; Liesegang, H.; Decker, I.; Herzberg, C.; Martinez Arias, R.; Merki, R.; Henne, A.; Gottshalk, G. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc. Natl. Acad. Sci. 2003, 100 (3), 1316 1321.

6. Functional Microbial Genomics; Wren, B., Dorrell, N., Eds.; Methods in Microbiology; Academic Press: London, UK, 2002; Vol. 33.

7. Rondon, M.R.; August, P.R.; Betterman, A.D.; Brady, S.F.; Grossman, T.H.; Liles, M.R.; Loiacono, K.A.; Lynch, B.A.; MacNeil, I.A.; Minor, C.; Tiong, C.L.; Gilman, M.; Osborne, M.S.; Clardy, J.; Handelsman, J.; Goodman, R.M. Cloning the soil metagenome: A strategy for accessing the genetic and functional diversity of uncultured micro organisms. Appl. Environ. Microbiol. 2000, 66 (6), 2541 2547.

8. Handfield, M.; Levesque, R.C. Strategies for isolation of in vivo expressed genes from bacteria. FEMS Microbiol. Rev. 1999, 23 (1), 69 91.

9. Vielle, C.; Zeikus, G.J. Hyperthermophilic enzymes: Sources, uses and molecular mechanisms for thermostabil ity. Microbiol. Mol. Biol. Rev. 2001, 65 (1), 1 43.

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