16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria.

Microorganisms play an important role in the biogeochemistry of the ocean surface layer, but spatial and temporal structures in the distributions of specific bacterioplankton species are largely unexplored, with the exceptions of those organisms that can be detected by either autofluorescence or culture methods. The use of rRNA genes as genetic markers provides a tool by which patterns in the growth, distribution, and activity of abundant bacterioplankton species can be studied regardless of the ease with which they can be cultured. Here we report an unusual cluster of related 16S rRNA genes (SAR202, SAR263, SAR279, SAR287, SAR293, SAR307) cloned from seawater collected at 250 m in the Sargasso Sea in August 1991, when the water column was highly stratified and the deep chlorophyll maximum was located at a depth of 120 m. Phylogenetic analysis and an unusual 15-bp deletion confirmed that the genes were related to the Green Non-Sulfur phylum of the domain Bacteria. This is the first evidence that representatives of this phylum occur in the open ocean. Oligonucleotide probes were used to examine the distribution of the SAR202 gene cluster in vertical profiles (0-250 m) from the Atlantic and Pacific Oceans, and in discrete (monthly) time series (O and 200 m) (over 30 consecutive months in the Western Sargasso Sea. The data provide robust statistical support for the conclusion that the SAR202 gene cluster is proportionately most abundant at the lower boundary of the deep chlorophyll maximum (P = 2.33 x 10(-5)). These results suggest that previously unsuspected stratification of microbial populations may be a significant factor in the ecology of the ocean surface layer.

Genomic evolution during a 10,000-generation experiment with bacteria

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria

In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection. If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions. Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives. The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions. Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA. Stabilities of Domain I of this subunit are 15–25% lower in endosymbionts than in closely related free-living bacteria. By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage. Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.

The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets

A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.

Mutators and sex in bacteria: Conflict between adaptive strategies

Bacterial mutation rates can increase and produce genetic novelty, as shown by in vitro and in silico experiments. Despite the cost due to a heavy deleterious mutation load, mutator alleles, which increase the mutation rate, can spread in asexual populations during adaptation because they remain associated with the rare favorable mutations they generate. This indirect selection for a genetic system generating diversity (second-order selection) is expected to be highly sensitive to changes in the dynamics of adaptation. Here we show by a simulation approach that even rare genetic exchanges, such as bacterial conjugation or transformation, can dramatically reduce the selection of mutators. Moreover, drift or competition between the processes of mutation and recombination in the course of adaptation reveal how second-order selection is unable to optimize the rate of generation of novelty.

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97–100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp

Wolbachia are bacteria that live in the cells of various invertebrate species to which they cause a wide range of effects on physiology and reproduction. We investigated the effect of Wolbachia infection in the parasitic wasp, Asobara tabida Nees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected by Wolbachia. The removal of Wolbachia by antibiotic treatment had a totally unexpected effect—aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treated Asobara citri, a closely related species that does not harbor Wolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents) are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production and Wolbachia density in females. We conclude that Wolbachia is necessary for oogenesis in these A. tabida strains, and this association would seem to be the first example of a transition from facultative to obligatory symbiosis in arthropod–Wolbachia associations.

Accelerated evolution and Muller's rachet in endosymbiotic bacteria.

Many bacteria live only within animal cells and infect hosts through cytoplasmic inheritance. These endosymbiotic lineages show distinctive population structure, with small population size and effectively no recombination. As a result, endosymbionts are expected to accumulate mildly deleterious mutations. If these constitute a substantial proportion of new mutations, endosymbionts will show (i) faster sequence evolution and (ii) a possible shift in base composition reflecting mutational bias. Analyses of 16S rDNA of five independently derived endosymbiont clades show, in every case, faster evolution in endosymbionts than in free-living relatives. For aphid endosymbionts (genus Buchnera), coding genes exhibit accelerated evolution and unusually low ratios of synonymous to nonsynonymous substitutions compared to ratios for the same genes for enterics. This concentration of the rate increase in nonsynonymous substitutions is expected under the hypothesis of increased fixation of deleterious mutations. Polypeptides for all Buchnera genes analyzed have accumulated amino acids with codon families rich in A+T, supporting the hypothesis that substitutions are deleterious in terms of polypeptide function. These observations are best explained as the result of Muller's ratchet within small asexual populations, combined with mutational bias. In light of this explanation, two observations reported earlier for Buchnera, the apparent loss of a repair gene and the overproduction of a chaperonin, may reflect compensatory evolution. An alternative hypothesis, involving selection on genomic base composition, is contradicted by the observation that the speedup is concentrated at nonsynonymous sites.

cAMP-dependent SOS induction and mutagenesis in resting bacterial populations.

The inducible SOS system increases the survival of bacteria exposed to DNA-damaging agents by increasing the capacity of error-free and error-prone DNA repair systems. The inducible mutator effect is expected to contribute to the adaptation of bacterial populations to these adverse life conditions by increasing their genetic variability. The evolutionary impact of the SOS system would be even greater if it was also induced under conditions common in nature, such as in resting bacterial populations. The results presented here show that SOS induction and mutagenesis do occur in bacteria in aging colonies on agar plates. The observed SOS induction and mutagenesis are controlled by the LexA repressor and are RecA- and cAMP-dependent.