Discovery of the Novel Candidate Phylum “Poribacteria” in Marine Sponges

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “Poribacteria”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “Poribacteria”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.     

A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc)

A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.     

Isolation, Pure Culture, and Characterization of “Candidatus Arsenophonus arthropodicus,” an Intracellular Secondary Endosymbiont from the Hippoboscid Louse Fly Pseudolynchia canariensis

Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as “Candidatus Arsenophonus arthropodicus.” Phylogenetic analyses indicate that “Candidatus Arsenophonus arthropodicus” is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of “Candidatus Arsenophonus arthropodicus” in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.     

Evolutionary Relationships of “Candidatus Riesia spp.,” Endosymbiotic Enterobacteriaceae Living within Hematophagous Primate Lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “Candidatus Arsenophonus insecticola.” We propose that two additional species be added to the genus “Candidatus Riesia.” The new species proposed within “Candidatus Riesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

An assessment of terminology for intraspecific diversity in fishes,with a focus on “ecotypes” and “life histories”

Understanding and preserving intraspecific diversity (ISD) is important for species conservation. However, ISD units do not have taxonomic standards and are not universally recognized. The terminology used to describe ISD is varied and often used ambiguously. We compared definitions of terms used to describe ISD with use in recent studies of three fish taxa: sticklebacks (Gasterosteidae), Pacific salmon and trout (Oncorhynchus spp., “PST”), and lampreys (Petromyzontiformes). Life history describes the phenotypic responses of organisms to environments and includes biological parameters that affect population growth or decline. Life‐history pathway(s) are the result of different organismal routes of development that can result in different life histories. These terms can be used to describe recognizable life‐history traits. Life history is generally used in organismal‐ and ecology‐based journals. The terms paired species/species pairs have been used to describe two different phenotypes, whereas in some species and situations a continuum of phenotypes may be expressed. Our review revealed overlapping definitions for race and subspecies, and subspecies and ecotypes. Ecotypes are genotypic adaptations to particular environments, and this term is often used in genetic‐ and evolution‐based journals. “Satellite species” is used for situations in which a parasitic lamprey yields two or more derived, nonparasitic lamprey species. Designatable Units, Evolutionary Significant Units (ESUs), and Distinct Population Segments (DPS) are used by some governments to classify ISD of vertebrate species within distinct and evolutionary significant criteria. In situations where the genetic or life‐history components of ISD are not well understood, a conservative approach would be to call them phenotypes.

The terminology used to describe intraspecific diversity is varied and often used ambiguously. “Ecotype” was originally used to describe patterns in genes and ecology, and recent studies employing this term tend to report a genetic basis in ISD. By contrast, “life history” describes biological parameters that affect demography, and this term tends to be used in organismal‐ and ecology‐based journals.     

Phylogenetic Characterization and Prevalence of “Spirobacillus cienkowskii,” a Red-Pigmented, Spiral-Shaped Bacterial Pathogen of Freshwater Daphnia Species

Microscopic examination of the hemolymph from diseased daphniids in 17 lakes in southwestern Michigan and five rock pools in southern Finland revealed the presence of tightly coiled bacteria that bore striking similarities to the drawings of a morphologically unique pathogen, “Spirobacillus cienkowskii,” first described by Elya Metchnikoff more than 100 years ago. The uncultivated microbe was identified as a deeply branching member of the Deltaproteobacteria through phylogenetic analyses of two conserved genes: the 16S rRNA-encoding gene (rrs) and the β-subunit of topoisomerase (gyrB). Fluorescence in situ hybridization confirmed that the rRNA gene sequence originated from bacteria with the tightly coiled morphology. Microscopy and PCR amplification with pathogen-specific primers confirmed infections by this bacterium in four species of Daphnia: Daphnia dentifera, D. magna, D. pulicaria, and D. retrocurva. Extensive field surveys reveal that this bacterium is widespread geographically and able to infect many different cladoceran species. In a survey of populations of D. dentifera in lakes in Michigan, we found the bacterium in 17 of 18 populations studied. In these populations, 0 to 12% of the individuals were infected, with an average of 3% during mid-summer and early autumn. Infections were less common in rock pool populations of D. magna in southern Finland, where the pathogen was found in 5 of 137 populations. The broad geographic distribution, wide host range, and high virulence of S. cienkowskii suggest it plays an important role in the ecology and evolution of daphniids.     

Cloning of the spoT Gene of “Candidatus Phlomobacter fragariae” and Development of a PCR-Restriction Fragment Length Polymorphism Assay for Detection of the Bacterium in Insects

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium “Candidatus Phlomobacter fragariae” is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between “P. fragariae” and other insect-associated proteobacteria, isolation of “P. fragariae” genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from “P. fragariae”-infected strawberry plants. It encodes part of a “P. fragariae” open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish “P. fragariae” from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry “P. fragariae.” Interestingly however, the “P. fragariae” spoT sequence could be easily detected in whiteflies proliferating on “P. fragariae”-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.     

Effect of Iron Limitation on “Pseudomonas plantarii” Growth and Tropolone and Protein Production

The addition of iron to an iron-deficient medium markedly enhanced the growth of “Pseudomonas plantarii” and the production of red crystals. However, it markedly reduced the amount of dissolved tropolone, a product of the bacterium and an iron chelator, and the production of an iron-regulated protein (78 kilodaltons). The red crystal was complex, composed of tropolone and iron, the ratio being 3:1.     

Response to “Vast (but avoidable) underestimation of global biodiversity”

This Formal Comment provides clarifications on the authors’ recent estimates of global bacterial diversity and the current status of the field, and responds to a Formal Comment from John Wiens regarding their prior work.

We welcome Wiens’ efforts to estimate global animal-associated bacterial richness and thank him for highlighting points of confusion and potential caveats in our previous work on the topic [1]. We find Wiens’ ideas worthy of consideration, as most of them represent a step in the right direction, and we encourage lively scientific discourse for the advancement of knowledge. Time will ultimately reveal which estimates, and underlying assumptions, came closest to the true bacterial richness; we are excited and confident that this will happen in the near future thanks to rapidly increasing sequencing capabilities. Here, we provide some clarifications on our work, its relation to Wiens’ estimates, and the current status of the field.First, Wiens states that we excluded animal-associated bacterial species in our global estimates. However, thousands of animal-associated samples were included in our analysis, and this was clearly stated in our main text (second paragraph on page 3).Second, Wiens’ commentary focuses on “S1 Text” of our paper [1], which was rather peripheral, and, hence, in the Supporting information. S1 Text [1] critically evaluated the rationale underlying previous estimates of global bacterial operational taxonomic unit (OTU) richness by Larsen and colleagues [2], but the results of S1 Text [1] did not in any way flow into the analyses presented in our main article. Indeed, our estimates of global bacterial (and archaeal) richness, discussed in our main article, are based on 7 alternative well-established estimation methods founded on concrete statistical models, each developed specifically for richness estimates from multiple survey data. We applied these methods to >34,000 samples from >490 studies including from, but not restricted to, animal microbiomes, to arrive at our global estimates, independently of the discussion in S1 Text [1].Third, Wiens’ commentary can yield the impression that we proposed that there are only 40,100 animal-associated bacterial OTUs and that Cephalotes in particular only have 40 associated bacterial OTUs. However, these numbers, mentioned in our S1 Text [1], were not meant to be taken as proposed point estimates for animal-associated OTU richness, and we believe that this was clear from our text. Instead, these numbers were meant as examples to demonstrate how strongly the estimates of animal-associated bacterial richness by Larsen and colleagues [2] would decrease simply by (a) using better justified mathematical formulas, i.e., with the same input data as used by Larsen and colleagues [2] but founded on an actual statistical model; (b) accounting for even minor overlaps in the OTUs associated with different animal genera; and/or (c) using alternative animal diversity estimates published by others [3], rather than those proposed by Larsen and colleagues [2]. Specifically, regarding (b), Larsen and colleagues [2] (pages 233 and 259) performed pairwise host species comparisons within various insect genera (for example, within the Cephalotes) to estimate on average how many bacterial OTUs were unique to each host species, then multiplied that estimate with their estimated number of animal species to determine the global animal-associated bacterial richness. However, since their pairwise host species comparisons were restricted to congeneric species, their estimated number of unique OTUs per host species does not account for potential overlaps between different host genera. Indeed, even if an OTU is only found “in one” Cephalotes species, it might not be truly unique to that host species if it is also present in members of other host genera. To clarify, we did not claim that all animal genera can share bacterial OTUs, but instead considered the implications of some average microbiome overlap (some animal genera might share no bacteria, and other genera might share a lot). The average microbiome overlap of 0.1% (when clustering bacterial 16S sequences into OTUs at 97% similarity) between animal genera used in our illustrative example in S1 Text [1] is of course speculative, but it is not unreasonable (see our next point). A zero overlap (implicitly assumed by Larsen and colleagues [2]) is almost certainly wrong. One goal of our S1 Text [1] was to point out the dramatic effects of such overlaps on animal-associated bacterial richness estimates using “basic” mathematical arguments.Fourth, Wiens’ commentary could yield the impression that existing data are able to tell us with sufficient certainty when a bacterial OTU is “unique” to a specific animal taxon. However, so far, the microbiomes of only a minuscule fraction of animal species have been surveyed. One can thus certainly not exclude the possibility that many bacterial OTUs currently thought to be “unique” to a certain animal taxon are eventually also found in other (potentially distantly related) animal taxa, for example, due to similar host diets and or environmental conditions [4–7]. As a case in point, many bacteria in herbivorous fish guts were found to be closely related to bacteria in mammals [8], and Song and colleagues [6] report that bat microbiomes closely resemble those of birds. The gut microbiome of caterpillars consists mostly of dietary and environmental bacteria and is not species specific [4]. Even in animal taxa with characteristic microbiota, there is a documented overlap across host species and genera. For example, there are a small number of bacteria consistently and specifically associated with bees, but these are found across bee genera at the level of the 99.5% similar 16S rRNA OTUs [5]. To further illustrate that an average microbiome overlap between animal taxa at least as large as the one considered in our S1 Text (0.1%) [1] is not unreasonable, we analyzed 16S rRNA sequences from the Earth Microbiome Project [6,9] and measured the overlap of microbiota originating from individuals of different animal taxa. We found that, on average, 2 individuals from different host classes (e.g., 1 mammalian and 1 avian sample) share 1.26% of their OTUs (16S clustered at 100% similarity), and 2 individuals from different host genera belonging to the same class (e.g., 2 mammalian samples) share 2.84% of their OTUs (methods in S1 Text of this response). A coarser OTU threshold (e.g., 97% similarity, considered in our original paper [1]) would further increase these average overlaps. While less is known about insect microbiomes, there is currently little reason to expect a drastically different picture there, and, as explained in our S1 Text [1], even a small average microbiome overlap of 0.1% between host genera would strongly limit total bacterial richness estimates. The fact that the accumulation curve of detected bacterial OTUs over sampled insect species does not yet strongly level off says little about where the accumulation curve would asymptotically converge; rigorous statistical methods, such as the ones used for our global estimates [1], would be needed to estimate this asymptote.Lastly, we stress that while the present conversation (including previous estimates by Louca and colleagues [1], Larsen and colleagues [2], Locey and colleagues [10], Wiens’ commentary, and this response) focuses on 16S rRNA OTUs, it may well be that at finer phylogenetic resolutions, e.g., at bacterial strain level, host specificity and bacterial richness are substantially higher. In particular, future whole-genome sequencing surveys may well reveal the existence of far more genomic clusters and ecotypes than 16S-based OTUs.     

Ultrastructure of Thiothrix spp. and “Type 021N” Bacteria

The ultrastructural features of two groups of filamentous sulfur bacteria, Thiothrix spp. and an unnamed organism designated “type 021N,” were examined by transmission electron microscopy. Negative staining of whole cells and filaments with uranyl acetate revealed the presence of tufts of fimbriae located at the ends of individual gonidia of Thiothrix sp. strain A1 and “type 021N” strain N7. Holdfast material present at the center of mature rosettes was observed in thin sections stained with ruthenium red. A clearly defined sheath enveloped the trichomes of two of three Thiothrix strains but was absent from “type 021N” filaments. The outer cell wall appeared more complex in “type 021N” strains than in Thiothrix isolates. Bulbs or clusters of irregularly shaped cells, often present in filaments of “type 021N” bacteria, appeared to result from crosswalls which formed at angles oblique to the filament axis. The multicellular nature of these sulfur bacteria was apparent in that only the cytoplasmic membrane and peptidoglycan layer of the cell wall were involved in the septation process. Sulfur inclusions which developed in the presence of sodium thiosulfate were enclosed by a single-layered envelope and located within invaginations of the cytoplasmic membrane.     

“Candidatus Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, “Candidatus Endobugula sertula.” “Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Evaluation of an adapted version of the Diabetes Prevention Program for low- and middle-income countries: A cluster randomized trial to evaluate “Lifestyle Africa” in South Africa

BackgroundLow- and middle-income countries (LMICs) are experiencing major increases in diabetes and cardiovascular conditions linked to overweight and obesity. Lifestyle interventions such as the United States National Diabetes Prevention Program (DPP) developed in high-income countries require adaptation and cultural tailoring for LMICs. The objective of this study was to evaluate the efficacy of “Lifestyle Africa,” an adapted version of the DPP tailored for an underresourced community in South Africa compared to usual care.Methods and findingsParticipants were residents of a predominantly Xhosa-speaking urban township of Cape Town, South Africa characterized by high rates of poverty. Participants with body mass index (BMI) ≥ 25 kg/m² who were members of existing social support groups or “clubs” receiving health services from local nongovernmental organizations (NGOs) were enrolled in a cluster randomized controlled trial that compared Lifestyle Africa (the intervention condition) to usual care (the control condition). The Lifestyle Africa intervention consisted of 17 video-based group sessions delivered by trained community health workers (CHWs). Clusters were randomized using a numbered list of the CHWs and their assigned clubs based on a computer-based random allocation scheme. CHWs, participants, and research team members could not be blinded to condition. Percentage weight loss (primary outcome), hemoglobin A1c (HbA1c), blood pressure, triglycerides, and low-density lipoprotein (LDL) cholesterol were assessed 7 to 9 months after enrollment. An individual-level intention-to-treat analysis was conducted adjusting for clustering within clubs and baseline values. Trial registration is at ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT03342274","term_id":"NCT03342274"}}NCT03342274). Between February 2018 and May 2019, 782 individuals were screened, and 494 were enrolled. Participants were predominantly retired (57% were receiving a pension) and female (89%) with a mean age of 68 years. Participants from 28 clusters were allocated to Lifestyle Africa (15, n = 240) or usual care (13, n = 254). Fidelity assessments indicated that the intervention was generally delivered as intended. The modal number of sessions held across all clubs was 17, and the mean attendance of participants across all sessions was 61%. Outcome assessment was completed by 215 (90%) intervention and 223 (88%) control participants. Intent-to-treat analyses utilizing multilevel modeling included all randomized participants. Mean weight change (primary outcome) was −0.61% (95% confidence interval (CI) = −1.22, −0.01) in Lifestyle Africa and −0.44% (95% CI = −1.06, 0.18) in control with no significant difference (group difference = −0.17%; 95% CI = −1.04, 0.71; p = 0.71). However, HbA1c was significantly lower at follow-up in Lifestyle Africa compared to the usual care group (mean difference = −0.24, 95% CI = −0.39, −0.09, p = 0.001). None of the other secondary outcomes differed at follow-up: systolic blood pressure (group difference = −1.36; 95% CI = −6.92, 4.21; p = 0.63), diastolic blood pressure (group difference = −0.39; 95% CI = −3.25, 2.30; p = 0.78), LDL (group difference = −0.07; 95% CI = −0.19, 0.05; p = 0.26), triglycerides (group difference = −0.02; 95% CI = −0.20, 0.16; p = 0.80). There were no unanticipated problems and serious adverse events were rare, unrelated to the intervention, and similar across groups (11 in Lifestyle Africa versus 13 in usual care). Limitations of the study include the lack of a rigorous dietary intake measure and the high representation of older women.ConclusionsIn this study, we found that Lifestyle Africa was feasible for CHWs to deliver and, although it had no effect on the primary outcome of weight loss or secondary outcomes of blood pressure or triglycerides, it had an apparent small significant effect on HbA1c. The study demonstrates the potential feasibility of CHWs to deliver a program without expert involvement by utilizing video-based sessions. The intervention may hold promise for addressing cardiovascular disease (CVD) and diabetes at scale in LMICs.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03342274","term_id":"NCT03342274"}}NCT03342274.

In a cluster randomized trial, Delwyn Catley and colleagues evaluate an adapted version of the Diabetes Prevention Program in South Africa.     

“Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.     

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

Secretory proteins perform a variety of important “remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ~90 extracellular proteins were identified. Analysis of these proteins disclosed various “secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only ~50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.