Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes (x ‫؍‬ 5.4) became dominant, whereas populations with fewer rRNA genes (x ‫؍‬ 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities.Genes encoding the 5S, 16S, and 23S rRNAs are typically organized into an operon in members of the domain Bacteria. The copy number of rRNA operons per bacterial genome varies from 1 to as many as 15 (28). For example, the pathogenic bacteria Rickettsia prowazekii (2) and Mycoplasma pneumoniae (4) have one rRNA operon, while the enteric bacteria Escherichia coli (12) and Salmonella enterica serovar Typhimurium (1) each possess seven copies per genome. The greatest number of rRNA operons per genome known can be found among spore-forming bacteria isolated from soil; Bacillus subtilis (23) and Clostridium paradoxum (28) possess 10 and 15 copies, respectively. Several hypotheses have been proposed to explain the wide variation observed in rRNA operon copy number.It is generally assumed that multiple copies of rRNA operons in prokaryotic organisms are required to achieve high growth rates. However, the short doubling time observed for certain bacteria with a single rRNA operon (37) and the marginal impact of rRNA operon inactivation on maximal growth rate (8,27) suggest that the capacity for rapid growth is not the sole determinant of rRNA operon copy number. The number of transcripts that can be initiated at an rRNA operon promoter and the transcriptional rate of RNA polymerase set a maximum rate on the number of ribosomes that can be produced from a single rRNA operon. Calculations including promoter initiation efficiency and transcription rates indicate that one copy of the rRNA operon is insufficient to supply the number of ribosomes required to achieve maximal growth rates observed in E. coli (5).Given the high demand for rRNA transcription and the central role of rRNAs in the regulation of ribosome synthesis, it is conceivable that the number of rRNA operons may dictate the rapidity with which microbes can synthesize ribosomes and respond to favorable changes in growth conditions (8,...

The complexity of soil bacterial communities has thus far confounded effective measurement. However, with improved analytical methods, we show that the abundance distribution and total diversity can be deciphered. Reanalysis of reassociation kinetics for bacterial community DNA from pristine and metal-polluted soils showed that a power law best described the abundance distributions. More than one million distinct genomes occurred in the pristine soil, exceeding previous estimates by two orders of magnitude. Metal pollution reduced diversity more than 99.9%, revealing the highly toxic effect of metal contamination, especially for rare taxa.

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.Comparative analysis of complex microbial communities in natural environments has been hampered by the lack of effective ways to rapidly measure community diversity, composition, and structure. The shortcomings of methods that rely on cultivation are well known, and although DNA-based, cultureindependent techniques have provided new ways to examine the microbial world, the methods that are effective in community analysis are still quite limited in number and scope (23). Terminal restriction fragment (length polymorphism (T-RFLP or TRF) analysis is currently one of the most powerful methods in microbial ecology for rapidly comparing the diversity of bacterial DNA sequences amplified by PCR from environmental samples (19,23). The method relies on variation in the position of restriction sites among sequences and determination of the length of fluorescently labeled TRFs by high-resolution gel electrophoresis on automated DNA sequencers (1, 16). The method's many strengths include speed and high sample throughput, which enables replicated experiments with statistical analysis to be conducted. Highly precise fragment length determination is achieved by use of an automated DNA sequencer with internal size standards in every profile and provides numerical data of exceptional resolution. In theory, data from the method can also be compared wi...

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.Rapid analysis of diversity in complex microbial communities has remained an elusive but important goal in microbial ecology. Community diversity can be examined at several levels. The most simple analyses use DNA profiles (generated by PCR and sometimes followed by restriction digestion of amplification mixtures) to identify differences in the composition of communities. More refined approaches describe differences not only in community composition but also in community organization by measuring the number (richness) and relative abundance (structure or evenness) of species or phylotypes. The richness and evenness of biological communities reflect selective pressures that shape diversity within communities. Measuring these parameters is most useful when assessing treatment effects (e.g., physical disturbance, pollution, nutrient addition, predation, climate change, etc.) on community diversity. Diversity statistics can also indicate the ability of a community to recover from disturbance and utilize resources efficiently (4). An ideal method for analysis of diversity in complex microbial communities would enable the simultaneous measurement of composition, phylotype richness, and community structure. The method would be rapid and reproducible and would permit flexible sampling of the entire microbial community.Direct amplification of bacterial 16S rRNA genes from extracted soil DNA provides the most comprehensive and flexible means of sampling bacterial communities. Analysis of clone libraries of 16S rRNA genes amplified from different environments can pro...

Soil bacteria are important contributors to primary productivity and nutrient cycling in arid land ecosystems, and their populations may be greatly affected by changes in environmental conditions. In parallel studies, the composition of the total bacterial community and of members of the Acidobacterium division were assessed in arid grassland soils using terminal restriction fragment length polymorphism (TRF, also known as T-RFLP) analysis of 16S rRNA genes amplified from soil DNA. Bacterial communities associated with the rhizospheres of the native bunchgrasses Stipa hymenoides and Hilaria jamesii, the invading annual grass Bromus tectorum, and the interspaces colonized by cyanobacterial soil crusts were compared at three depths. When used in a replicated field-scale study, TRF analysis was useful for identifying broad-scale, consistent differences in the bacterial communities in different soil locations, over the natural microscale heterogeneity of the soil. The compositions of the total bacterial community and Acidobacterium division in the soil crust interspaces were significantly different from those of the plant rhizospheres. Major differences were also observed in the rhizospheres of the three plant species and were most apparent with analysis of the Acidobacterium division. The total bacterial community and the Acidobacterium division bacteria were affected by soil depth in both the interspaces and plant rhizospheres. This study provides a baseline for monitoring bacterial community structure and dynamics with changes in plant cover and environmental conditions in the arid grasslands.