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.

Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leacbate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.

Molecular investigation of the microbial populations of the pink sugarcane mealybug, Saccharicoccus sacchari

In an attempt to better understand the microbial diversity and endosymbiotic microbiota of the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homoptera: Pseudococcidae), culture-independent approaches, namely PCR, a 16S rDNA clone library, and temperature gradient gel electrophoresis (TGGE) were used. Previous work has indicated that the acetic acid bacteria Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens represent only a small proportion of the microbial community of the PSMB. These findings were supported in this study by TGGE, where no bands representing G. sacchari, G. diazotrophicus, and G. liquefaciens on the acrylamide gel could be observed following electrophoresis, and by a 16S rDNA clone library study, where no clones with the sequence of an acetic acid bacterium were found. Instead, TGGE revealed that the mealybug microbial community was dominated by beta- and gamma-Proteobacteria. The dominant band in TGGE gels found in a majority of the mealybug samples was most similar, according to BLAST analysis, to the beta-symbiont of the craw mealybug Antonina crawii and to Candidatus Tremblaya princeps, an endosymbiont from the mealybug Paracoccus nothofagicola. The sequences of other dominant bands were identified as gamma-Proteobacteria, and were most closely related to uncultured bacterial clones obtained from soil samples. Mealybugs collected from different areas in Queensland, Australia, were found to produce similar TGGE profiles, although there were a few exceptions. A 16S rDNA clone library based on DNA extracted from a mealybug collected from sugarcane in the Burdekin region in Queensland, Australia, indicated very low levels of diversity among mealybug microbial populations. All sequenced clones were most closely related to the same members of the gamma-Proteobacteria, according to BLAST analysis.

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-Lanaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm. (C) 2005 Wiley Periodicals, Inc.

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation' Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO2- uptake than NH4 uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO2- from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal. (c) 2006 Elsevier Inc. All rights reserved.

Changes in equine hindgut bacterial populations during oligofructose-induced laminitis

In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24-32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse.

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".

(Table 4) Distribution of bacterial populations in sediments of ODP Hole 128-798B

Sediment whole-round cores from a dedicated hole (798B) were obtained for detailed microbiological analysis, down to 518 m below the seafloor (mbsf). These sediments have characteristic bacterial profiles in the top 6 mbsf, with high but rapidly decreasing bacterial populations (total and dividing bacteria, and concentrations of different types of viable heterotrophic bacteria) and potential bacterial activities. Rates of thymidine incorporation into bacterial DNA and anaerobic sulfate reduction are high in the surface sediments and decrease rapidly down to 3 mbsf. Methanogenesis from CO2/H2 peaks below the maximum in sulfate reduction and although it decreases markedly down the core, is present at low rates at all but one depth. Consistent with these activities is the removal of pore-water sulfate, methane gas production, and accumulation of reduced sulfide species. Rates of decrease in bacterial populations slow down below 6 mbsf, and there are some distinct increases in bacterial populations and activities that continue over considerable depth intervals. These include a large and significant increase in total heterotrophic bacteria below 375 mbsf, which corresponds to an increase in the total bacterial population, bacterial viability, a small increase in potential rates of sulfate reduction, and the presence of thermogenic methane and other gases. Bacterial distributions seem to be controlled by the availability of terminal electron acceptors (e.g., sulfate), the bioavailability of organic carbon (which may be related to the dark/light bands within the sediment), and biological and geothermal methane production. Significant bacterial populations are present even in the deepest samples (518 mbsf) and hence it seems likely that bacteria may continue to be present and active much deeper than the sediments studied here. These results confirm and extend our previous results of bacterial activity within deep sediments of the Peru Margin from Leg 112, and to our knowledge this is the first comprehensive report of the presence of active bacterial populations from the sediment surface to in excess of 500 mbsf and sediments > 4 m.y. old.

Comparative genomic approaches for investigating evolution in the microbial and viral populations of the rumen

The rumen is home to a diverse population of microorganisms encompassing all three domains of life: Bacteria, Archaea, and Eukarya. Viruses have also been documented to be present in large numbers; however, little is currently known about their role in the dynamics of the rumen ecosystem. This research aimed to use a comparative genomics approach in order to assess the potential evolutionary mechanisms at work in the rumen environment. We proposed to do this by first assessing the diversity and potential for horizontal gene transfer (HGT) of multiple strains of the cellulolytic rumen bacterium, Ruminococcus flavefaciens, and then by conducting a survey of rumen viral metagenome (virome) and subsequent comparison of the virome and microbiome sequences to ascertain if there was genetic information shared between these populations. We hypothesize that the bacteriophages play an integral role in the community dynamics of the rumen, as well as driving the evolution of the rumen microbiome through HGT. In our analysis of the Ruminococcus flavefaciens genomes, there were several mobile elements and clustered regularly interspaced short palindromic repeat (CRISPR) sequences detected, both of which indicate interactions with bacteriophages. The rumen virome sequences revealed a great deal of diversity in the viral populations. Additionally, the microbial and viral populations appeared to be closely associated; the dominant viral types were those that infect the dominant microbial phyla. The correlation between the distribution of taxa in the microbiome and virome sequences as well as the presence of CRISPR loci in the R. flavefaciens genomes, suggested that there is a “kill-the-winner” community dynamic between the viral and microbial populations in the rumen. Additionally, upon comparison of the rumen microbiome and rumen virome sequences, we found that there are many sequence similarities between these populations indicating a potential for phage-mediated HGT. These results suggest that the phages represent a gene pool in the rumen that could potentially contain genes that are important for adaptation and survival in the rumen environment, as well as serving as a molecular ‘fingerprint’ of the rumen ecosystem.