From genome mining to phenotypic microarrays: Planctomycetes as source for novel bioactive molecules

Most members of the phylum Planctomycetes share many unusual traits that are unique for bacteria, since they divide independent of FtsZ through asymmetric budding, possess a complex life cycle and comprise a compartmentalized cell plan. Besides their complex cell biological features Planctomycetes are environmentally important and play major roles in global matter fluxes. Such features have been successfully employed in biotechnological applications such as the anaerobic oxidation of ammonium in wastewater treatment plants or the utilization of enzymes for biotechnological processes. However, little is known about planctomycetal secondary metabolites. This is surprising as Planctomycetes have several key features in common with known producers of small bioactive molecules such as Streptomycetes or Myxobacteria: a complex life style and large genome sizes. Planctomycetal genomes with an average size of 6.9 MB appear as tempting targets for drug discovery approaches. To enable the hunt for bioactive molecules from Planctomycetes, we performed a comprehensive genome mining approach employing the antiSMASH secondary metabolite identification pipeline and found 102 candidate genes or clusters within the analyzed 13 genomes. However, as most genes and operons related to secondary metabolite production are exclusively expressed under certain environmental conditions, we optimized Phenotype MicroArray protocols for Rhodopirellula baltica and Planctomyces limnophilus to allow high throughput screening of putative stimulating carbon sources. Our results point towards a previously postulated relationship of Planctomycetes with algae or plants, which secrete compounds that might serve as trigger to stimulate the secondary metabolite production in Planctomycetes. Thus, this study provides the necessary starting point to explore planctomycetal small molecules for drug development.     

A Unique Pool of Compatible Solutes on Rhodopirellula baltica,Member of the Deep-Branching Phylum Planctomycetes

The intracellular accumulation of small organic solutes was described in the marine bacterium Rhodopirellula baltica, which belongs to the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. Sucrose, α-glutamate, trehalose and mannosylglucosylglycerate (MGG) are the main solutes involved in the osmoadaptation of R. baltica. The ratio and total intracellular organic solutes varied significantly in response to an increase in salinity, temperature and nitrogen content. R. baltica displayed an initial response to both osmotic and thermal stresses that includes α-glutamate accumulation. This trend was followed by a rather unique and complex osmoadaptation mechanism characterized by a dual response to sub-optimal and supra-optimal salinities. A reduction in the salinity to sub-optimal conditions led primarily to the accumulation of trehalose. In contrast, R. baltica responded to salt stress mostly by increasing the intracellular levels of sucrose. The switch between the accumulation of trehalose and sucrose was by far the most significant effect caused by increasing the salt levels of the medium. Additionally, MGG accumulation was found to be salt- as well as nitrogen-dependent. MGG accumulation was regulated by nitrogen levels replacing α-glutamate as a K⁺ counterion in nitrogen-poor environments. This is the first report of the accumulation of compatible solutes in the phylum Planctomycetes and of the MGG accumulation in a mesophilic organism.     

Whole-Genome Reciprocal BLAST Analysis Reveals that Planctomycetes Do Not Share an Unusually Large Number of Genes with Eukarya and Archaea

The genome sequences of Rhodopirellula baltica, formerly Pirellula sp. strain 1, Blastopirellula marina, Gemmata obscuriglobus, and Kuenenia stuttgartiensis were used in a series of pairwise reciprocal best-hit analyses to evaluate the contested evolutionary position of Planctomycetes. Contrary to previous reports which suggested that R. baltica had a high percentage of genes with closest matches to Archaea and Eukarya, we show here that these Planctomycetes do not share an unusually large number of genes with the Archaea or Eukarya, compared with other Bacteria. Thus, best-hit analyses may assign phylogenetic affinities incorrectly if close relatives are absent from the sequence database.     

Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes

Background  

The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization.     

The complete chloroplast and mitochondrial genomes of the diatom Nitzschia palea (Bacillariophyceae) demonstrate high sequence similarity to the endosymbiont organelles of the dinotom Durinskia baltica

Nitzschia palea is a common freshwater diatom used as a bioindicator because of its tolerance of polluted waterways. There is also evidence it may be the tertiary endosymbiont within the “dinotom” dinoflagellate Durinskia baltica. A putative strain of N. palea was collected from a pond on the University of Virginia's College at Wise campus and cultured. For initial identification, three markers were sequenced—nuclear 18S rDNA, the chloroplast 23S rDNA, and rbcL. Morphological characteristics were determined using light and scanning electron microscopy; based on these observations the cells were identified as N. palea and named strain “Wise.” DNA from N. palea was deep sequenced and the chloroplast and mitochondrial genomes assembled. Single gene phylogenies grouped N. palea—Wise within a clearly defined N. palea clade and showed it was most closely related to the strain “SpainA3.” The chloroplast genome of N. palea is 119,447 bp with a quadripartite structure, 135 protein‐coding, 28 tRNA, and 3 rRNA genes. The mitochondrial genome is 37,754 bp with a single repeat region as found in other diatom chondriomes, 37 protein‐coding, 23 tRNA, and 2 rRNA genes. The chloroplast genomes of N. palea and D. baltica have identical gene content, synteny, and a 92.7% pair‐wise sequence similarity with most differences occurring in intergenic regions. The N. palea mitochondrial genome and D. baltica's endosymbiont mitochondrial genome also have identical gene content and order with a sequence similarity of 90.7%. Genome‐based phylogenies demonstrated that D. baltica is more similar to N. palea than any other diatom sequence currently available. These data provide the genome sequences of two organelles for a widespread diatom and show they are very similar to those of Durinskia baltica's endosymbiont.     

Genetic diversity of Rhodopirellula strains

Rhodopirellula baltica SH1^T is a marine planctomycete with 7,325 genes in its genome. Ten strains of the genus Rhodopirellula were studied in whole genome microarray experiments to assess the extent of their genetic relatedness to R. baltica SH1^T. DNA of strains which were previously affiliated with the species R. baltica (OTU A) hybridized with 3,645–5,728 genes of the type strain on the microarray. Strains SH398 and 6C (OTU B), representing a closely related species with an average nucleotide identity of 88 %, showed less hybridization signals: 1,816 and 3,302 genes gave a hybridization signal, respectively. Comparative genomics of eight permanent draft genomes revealed the presence of over 4,000 proteins common in R. baltica SH1^T and strains of OTU A or B. The genus Rhodopirellula is characterized by large genomes, with over 7,000 genes per genome and a core genome of around 3000 genes. Individual Rhodopirellula strains have a large portion of strain-specific genes.     

Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems

The cell cycle of Caulobacter crescentus is controlled by a complex signalling network that co‐ordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non‐essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism.     

In this issue: Proteomics 8/2008

In this issue of Proteomics you will find the following highlighted articles: Have a heart (mitochondrial) proteome Is a rose always a rose? How clean is clean? Is a proteome always a proteome? Such deep questions to ponder. Zhang et al. don't just ponder, they attack the last two questions. Taking meticulous care to prepare clean mouse cardiac mitochondria, they identify almost a thousand proteins from the functionally and morphologically validated organelle. Half of the proteins had not been previously identified. Functional clusters include the expected and the “under‐appreciated” – proteolysis, protein folding, apoptosis and redox signaling. A close association with rough ER could not be disrupted without damage to the outer mitochondrial membrane. Immunocytological localization of many of the proteins revealed roles in other sites as well, including ER, cytoplasm, and Golgi. Comparative analysis of published mitochondrial proteomes from different tissues suggests that the proteomes are functionally adapted to their particular milieu. A mitochondrion (heart) is not a mitochondrion (liver). Zhang, J. et al., Proteomics 2008, 8, 1564–1575. Ibuprofen: split personality complicates proteome analyses Ibuprofen is one of those two‐fisted drugs that comes in an S form and an R form. The S form of this nonsteroidal anti‐inflammatory drug (NSAID) is the only active one, in this case. Normally sold over the counter for general aches and pains in the US, statistical analysis of its regular users has found it associated with a reduced incidence of Alzheimer's disease. Following up on this lead, Zhang et al. performed proteomic analysis of the effect of the R and S forms and their mixture on neuroblastoma cells. From three replicates, 167 proteins were identified as being quantitatively shifted. A total of 13 were unique. Functionally, they included representatives from metabolic enzymes (5), signaling (6), and cytoskeleton (2). Of interest for the Alzheimer's association was the reduced levels of reactive oxygen species (ROS), probably linked to levels of peroxiredoxins 2 and 6 in ibuprofen S‐treated cells. Zhang, J. et al., Proteomics 2008, 8, 1595–1607. Not your usual marine bacterium Rhodopirellula baltica is a member of the Planctomycetes phylum. These bacteria exhibit a proteinaceous cell wall, budding cell division, and intracellular compartments. From genome sequencing, it has >7300 ORFs. Analyzing the soluble proteins over the range of pH 3–10 by 2‐D PAGE, using narrow range pH gradient gels, nHPLC‐MS, and 1‐D SDS‐PAGE, Hieu et al. added 709 proteins to the proteins identified previously to bring the total identified to 1267, 17% of the predicted total ORFs. Gel‐free analysis (multiple dimension LC‐MS) yielded 145 proteins not seen in gel‐based methods. Both 1‐D and gel‐free methods were used for identification of cell wall and ribosomal proteins. Ninety three proteins were identified in the cell wall proteome and 13 extracellular proteins. No support was found for the hypothesis that R. baltica fed on sinking dead “marine snow” organisms by secreting proteases. Hieu, C. X. et al., Proteomics 2008, 8, 1608–1623.     

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control

Background

Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism.

Results

We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio.

Conclusions

Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.     

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

Background  

Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy.     

Cover Picture: Proteomics 8/2008

In this issue of Proteomics you will find the following highlighted articles: Have a heart (mitochondrial) proteome Is a rose always a rose? How clean is clean? Is a proteome always a proteome? Such deep questions to ponder. Zhang et al. don't just ponder, they attack the last two questions. Taking meticulous care to prepare clean mouse cardiac mitochondria, they identify almost a thousand proteins from the functionally and morphologically validated organelle. Half of the proteins had not been previously identified. Functional clusters include the expected and the “under‐appreciated” – proteolysis, protein folding, apoptosis and redox signaling. A close association with rough ER could not be disrupted without damage to the outer mitochondrial membrane. Immunocytological localization of many of the proteins revealed roles in other sites as well, including ER, cytoplasm, and Golgi. Comparative analysis of published mitochondrial proteomes from different tissues suggests that the proteomes are functionally adapted to their particular milieu. A mitochondrion (heart) is not a mitochondrion (liver). Zhang, J. et al., Proteomics 2008, 8, 1564–1575. Ibuprofen: split personality complicates proteome analyses Ibuprofen is one of those two‐fisted drugs that comes in an S form and an R form. The S form of this nonsteroidal anti‐inflammatory drug (NSAID) is the only active one, in this case. Normally sold over the counter for general aches and pains in the US, statistical analysis of its regular users has found it associated with a reduced incidence of Alzheimer's disease. Following up on this lead, Zhang et al. performed proteomic analysis of the effect of the R and S forms and their mixture on neuroblastoma cells. From three replicates, 167 proteins were identified as being quantitatively shifted. A total of 13 were unique. Functionally, they included representatives from metabolic enzymes (5), signaling (6), and cytoskeleton (2). Of interest for the Alzheimer's association was the reduced levels of reactive oxygen species (ROS), probably linked to levels of peroxiredoxins 2 and 6 in ibuprofen S‐treated cells. Zhang, J. et al., Proteomics 2008, 8, 1595–1607. Not your usual marine bacterium Rhodopirellula baltica is a member of the Planctomycetes phylum. These bacteria exhibit a proteinaceous cell wall, budding cell division, and intracellular compartments. From genome sequencing, it has >7300 ORFs. Analyzing the soluble proteins over the range of pH 3–10 by 2‐D PAGE, using narrow range pH gradient gels, nHPLC‐MS, and 1‐D SDS‐PAGE, Hieu et al. added 709 proteins to the proteins identified previously to bring the total identified to 1267, 17% of the predicted total ORFs. Gel‐free analysis (multiple dimension LC‐MS) yielded 145 proteins not seen in gel‐based methods. Both 1‐D and gel‐free methods were used for identification of cell wall and ribosomal proteins. Ninety three proteins were identified in the cell wall proteome and 13 extracellular proteins. No support was found for the hypothesis that R. baltica fed on sinking dead “marine snow” organisms by secreting proteases. Hieu, C. X. et al., Proteomics 2008, 8, 1608–1623.     

Wide distribution of Phycisphaera-like planctomycetes from WD2101 soil group in peatlands and genome analysis of the first cultivated representative

Phycisphaera-like WD2101 ‘soil group’ is one of the as-yet-uncultivated phylogenetic clades within the phylum Planctomycetes. Members of this clade are commonly detected in various terrestrial habitats. This study shows that WD2101 represented one of the major planctomycete groups in 10 boreal peatlands, comprising up to 76% and 36% of all Planctomycetes-affiliated 16S rRNA gene reads in raised bogs and eutrophic fens respectively. These types of peatlands displayed clearly distinct intra-group diversity of WD2101-affiliated planctomycetes. The first isolate of this enigmatic planctomycete group, strain M1803, was obtained from a humic lake surrounded by Sphagnum peat bogs. Strain M1803 displayed 89.2% 16S rRNA gene similarity to Tepidisphaera mucosa and was represented by motile cocci that divided by binary fission and grew under micro-oxic conditions. The complete 7.19 Mb genome of strain M1803 contained an array of genes encoding Planctomycetal type bacterial microcompartment organelle likely involved in l -rhamnose metabolism, suggesting participation of M1803-like planctomycetes in polysaccharide degradation in peatlands. The corresponding cellular microcompartments were revealed in ultrathin cell sections. Strain M1803 was classified as a novel genus and species, Humisphaera borealis gen. nov., sp. nov., affiliated with the formerly recognized WD2101 ‘soil group’.     

Archaea-Like Genes for C<Subscript>1</Subscript>-<Emphasis Type="Bold">Transfer Enzymes</Emphasis> in <Emphasis Type="Italic">Planctomycetes</Emphasis>: Phylogenetic Implications of Their Unexpected Presence in This Phylum

The unexpected presence of archaea-like genes for tetrahydromethanopterin (H₄MPT)-dependent enzymes in the completely sequenced genome of the aerobic marine planctomycete Pirellula sp. strain 1 (Rhodopirellula baltica) and in the currently sequenced genome of the aerobic freshwater planctomycete Gemmata obscuriglobus strain UQM2246 revives the discussion on the origin of these genes in the bacterial domain. We compared the genomic arrangement of these genes in Planctomycetes and methylotrophic proteobacteria and performed a phylogenetic analysis of the encoded protein sequences to address the question whether the genes have been present in the common ancestor of Bacteria and Archaea or were transferred laterally from the archaeal to the bacterial domain and therein. Although this question could not be solved using the data presented here, some constraints on the evolution of the genes involved in archaeal and bacterial H₄MPT-dependent C₁-transfer may be proposed: (i) lateral gene transfer (LGT) from Archaea to a common ancestor of Proteobacteria and Planctomycetes seems more likely than the presence of the genes in the common ancestor of Bacteria and Archaea; (ii) a single event of interdomain LGT can be favored over two independent events; and (iii) the archaeal donor of the genes might have been a representative of the Methanosarcinales. In the bacterial domain, the acquired genes evolved according to distinct environmental and metabolic constraints, reflected by specific rearrangements of gene order, gene recruitment, and gene duplication, with subsequent functional specialization. During the course of evolution, genes were lost from some planctomycete genomes or replaced by orthologous genes from proteobacterial lineages.Reviewing Editor: Dr. W. Ford Doolittle     

Genome Analysis of the Anaerobic Thermohalophilic Bacterium Halothermothrix orenii

Halothermothirx orenii is a strictly anaerobic thermohalophilic bacterium isolated from sediment of a Tunisian salt lake. It belongs to the order Halanaerobiales in the phylum Firmicutes. The complete sequence revealed that the genome consists of one circular chromosome of 2578146 bps encoding 2451 predicted genes. This is the first genome sequence of an organism belonging to the Haloanaerobiales. Features of both Gram positive and Gram negative bacteria were identified with the presence of both a sporulating mechanism typical of Firmicutes and a characteristic Gram negative lipopolysaccharide being the most prominent. Protein sequence analyses and metabolic reconstruction reveal a unique combination of strategies for thermophilic and halophilic adaptation. H. orenii can serve as a model organism for the study of the evolution of the Gram negative phenotype as well as the adaptation under thermohalophilic conditions and the development of biotechnological applications under conditions that require high temperatures and high salt concentrations.     

Identification of Proteins Likely To Be Involved in Morphogenesis,Cell Division,and Signal Transduction in Planctomycetes by Comparative Genomics

Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a “guilt-by-association” approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.     

Ammonium and attachment of <Emphasis Type="Italic">Rhodopirellula baltica</Emphasis>

A dimorphic life cycle has been described for the planctomycete Rhodopirellula baltica SH1^T, with juvenile motile, free-swimming cells and adult sessile, attached-living cells. However, attachment as a response to environmental factors was not investigated. We studied the response of R. baltica to nitrogen limitation. In batch cultures, ammonium limitation coincided with a dominance of free-swimming cells and a low number of aggregates. Flow cytometry revealed a quantitative shift with increasing ammonium availability, from single cells towards attached cells in large aggregates. During growth of R. baltica on glucose and ammonium in chemostats, an ammonium addition caused a macroscopic change of the growth behaviour, from homogeneous growth in the liquid phase to a biofilm on the borosilicate glass wall of the chemostat vessel. Thus, an ammonium limitation—a carbon to nitrogen supply ratio of 30:1—sustained free-living growth without aggregate formation. A sudden increase in ammonium supply induced sessile growth of R. baltica. These observations reveal a response of Rhodopirellula baltica cells to ammonium: they abandon the free-swimming life, attach to particles and form biofilms.     

The dinoflagellates <Emphasis Type="Italic">Durinskia baltica</Emphasis> and <Emphasis Type="Italic">Kryptoperidinium foliaceum</Emphasis> retain functionally overlapping mitochondria from two evolutionarily distinct lineages

Background  

The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum are distinguished by the presence of a tertiary plastid derived from a diatom endosymbiont. The diatom is fully integrated with the host cell cycle and is so altered in structure as to be difficult to recognize it as a diatom, and yet it retains a number of features normally lost in tertiary and secondary endosymbionts, most notably mitochondria. The dinoflagellate host is also reported to retain mitochondrion-like structures, making these cells unique in retaining two evolutionarily distinct mitochondria. This redundancy raises the question of whether the organelles share any functions in common or have distributed functions between them.