'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction

Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored 'Ca. Liberibacter asiaticus,' is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. Only two 'Ca. Liberibacter asiaticus' proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na(+)/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and 'Ca. Liberibacter asiaticus' orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤ E-10) with 'Ca. Liberibacter asiaticus' proteins, often present as multiple orthologs of single 'Ca. Liberibacter asiaticus' proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational analysis and is consistent with the hypothesis that these proteins may be of particular importance in host/microbe interaction and their duplication likely facilitates their ongoing evolution.     

'Ca. Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.     

Molecular characterization of a mosaic locus in the genome of '<Emphasis Type=Italic>Candidatus</Emphasis>Liberibacter asiaticus'

Background  

Huanglongbing (HLB) is a highly destructive disease of citrus production worldwide. 'Candidatus Liberibacter asiaticus', an unculturable alpha proteobacterium, is a putative pathogen of HLB. Information about the biology and strain diversity of 'Ca. L. asiaticus' is currently limited, inhibiting the scope of HLB research and control.     

Metagenomics approach for Polymyxa betae genome assembly enables comparative analysis towards deciphering the intracellular parasitic lifestyle of the plasmodiophorids

Genomic knowledge of the tree of life is biased to specific groups of organisms. For example, only six full genomes are currently available in the rhizaria clade. Here, we have applied metagenomic techniques enabling the assembly of the genome of Polymyxa betae (Rhizaria, Plasmodiophorida) RES F41 isolate from unpurified zoospore holobiont and comparison with the A26–41 isolate. Furthermore, the first P. betae mitochondrial genome was assembled. The two P. betae nuclear genomes were highly similar, each with just ~10.2 k predicted protein coding genes, ~3% of which were unique to each isolate. Extending genomic comparisons revealed a greater overlap with Spongospora subterranea than with Plasmodiophora brassicae, including orthologs of the mammalian cation channel sperm-associated proteins, raising some intriguing questions about zoospore physiology. This work validates our metagenomics pipeline for eukaryote genome assembly from unpurified samples and enriches plasmodiophorid genomics; providing the first full annotation of the P. betae genome.     

A Rickettsia genome overrun by mobile genetic elements provides insight into the acquisition of genes characteristic of an obligate intracellular lifestyle

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ~35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.     

Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family.

The membrane-bound complex of periplasmic permeases comprises two hydrophobic proteins which have been hypothesized to be integral membrane-spaninning proteins. We have investigated the topological organization of the hydrophobic components of the Salmonella typhimurium histidine permease, HisQ and HisM. Both proteins are digested by trypsin and proteinase K when either inside-out or right-side-out membrane vesicles are used. Therefore, these proteins are exposed to both surfaces of the membrane. Digestion with carboxypeptidase and binding studies with antibodies directed against the carboxyl terminus of HisQ and HisM have localized their carboxyl termini to the inside surface of the cytoplasmic membrane. Aminopeptidase digestion suggests periplasmic localization of their amino termini. Alkaline phosphatase fusions to HisQ and HisM indicate the existence of five spanners in both proteins. The periodicity and orientation of spanners and loops in HisQ and HisM match those of the five carboxyl-terminal spanners of MalF, the only other hydrophobic component of the periplasmic permeases for which topological information is available. An alignment of the sequences of all known hydrophobic components of periplasmic permeases is presented which indicates clear conservation of secondary structure and some conservation of primary sequence. The structural conservation of the components is discussed, and a role for a hydrophilic loop containing a conserved sequence (the EAA loop) is proposed.     

Comparative genome characterization of Achromobacter members reveals potential genetic determinants facilitating the adaptation to a pathogenic lifestyle

Members of the Achromobacter genus are Gram-negative bacteria including both environmental and clinical isolates, which are increasingly recovered from patients with cystic fibrosis (CF) as emerging pathogens. To better understand the features of the genus and its potential pathogenic mechanisms, six available Achromobacter genomes were compared in this study. The results revealed that: (1) Achromobacter had a pan-genome size of 10,750 genes with 3,398 core genes and a similar global classification of protein functions; (2) the Achromobacter genomes underwent a relatively low recombination that introduced nearly twice nucleotide substitutions less than the point mutation in genome evolution; (3) phylogenomic analysis based on 436 conserved proteins and average nucleotide identity both indicated that the Achromobacter genus had the closest relationship to the human/animal pathogen Bordetella rather than to Alcaligenes. The entire group of Achromobacter clustered with Bordetella in phylogeny, strongly suggesting a common origin, which therefore highlighted the potentially pathogenic nature of Achromobacter from the phylogenetic perspective, and (4) the CF clinical isolate possessed markedly unique genomic features discriminated from the environmental isolate and was equipped with numerous factors that facilitate its adaptation to a pathogenic lifestyle, such as a type III secretion system, a “polysaccharide island” (36.0 kb) of capsular/cellulose synthesis, adhesion-related proteins, alcaligin biogenesis, and several putative toxins. This study provided the first comprehensive genomic comparative analysis for Achromobacter, revealed information to better understand this far less-known genus on the genomic scale, and, importantly, identified potential virulence factors of the Achromobacter pathogen.     

Reduction of bacterial genome size and expansion resulting from obligate intracellular lifestyle and adaptation to soil habitat.

Prokaryotic organisms are exposed in the course of evolution to various impacts, resulting often in drastic changes of their genome size. Depending on circumstances, the same lineage may diverge into species having substantially reduced genomes, or such whose genomes have undergone considerable enlargement. Genome reduction is a consequence of obligate intracellular lifestyle rendering numerous genes expendable. Another consequence of intracellular lifestyle is reduction of effective population size and limited possibility of gene acquirement via lateral transfer. This causes a state of relaxed selection resulting in accumulation of mildly deleterious mutations that can not be corrected by recombination with the wild type copy. Thus, gene loss is usually irreversible. Additionally, constant environment of the eukaryotic cell renders that some bacterial genes involved in DNA repair are expandable. The loss of these genes is a probable cause of mutational bias resulting in a high A+T content. While causes of genome reduction are rather indisputable, those resulting in genome expansion seem to be less obvious. Presumably, the genome enlargement is an indirect consequence of adaptation to changing environmental conditions and requires the acquisition and integration of numerous genes. It seems that the need for a great number of capabilities is common among soil bacteria irrespective of their phylogenetic relationship. However, this would not be possible if soil bacteria lacked indigenous abilities to exchange and accumulate genetic information. The latter are considerably facilitated when housekeeping genes are physically separated from adaptive loci which are useful only in certain circumstances.     

Cytogenetic and molecular characterization of the Abies alba genome and its relationship with other members of the Pinaceae

Genome size, karyotype structure, heterochromatin distribution, position and number of ribosomal genes, as well as the ITS2 sequence of the internal transcribed spacer (ITS) were analysed in silver fir (Abies alba Mill.). The analysis also included characterization of the Arabidopsis-type of telomeric repeats in silver fir and in related species. The results were compared with results from other species of the Pinaceae, to evaluate phylogeny and chromosomal and molecular evolution in the Pinaceae. Integrated chromosomal data provided insights into chromosome and karyotype evolution in the Pinaceae. The evolutionary trend for GC-rich heterochromatic blocks seems to involve loss of blocks that are not associated with rDNA. Similarly, numerous large blocks of interstitial plant telomeric repeats that are typical for all analysed species of the genus Pinus were not observed in the evolutionarily younger genera, such as Abies, Picea and Larix. On the contrary, the majority of telomeric sequences in these three genera appeared confined to the chromosome ends. We confirmed the current position of Abies and Tsuga in subfamily Abietoideae and the position of Pinus in the subfamily Pinoideae based on ITS2 sequences. Pseudotsuga is placed together with Larix into the subfamily Laricoideae. We conclude that the current position of the genus Picea in the subfamily Abietoideae should be reconsidered and, possibly, the genus Picea should be reclassified as a separate subfamily, Piceoideae, as recently proposed.     

The complete genome sequence of 'Candidatus Liberibacter solanacearum', the bacterium associated with potato zebra chip disease

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with 'Candidatus Liberibacter solanacearum', a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for 'Ca. L. solanacearum'. Here we present the sequence of the 1.26 Mbp metagenome of 'Ca. L. solanacearum', based on DNA isolated from potato psyllids. The coding inventory of the 'Ca. L. solanacearum' genome was analyzed and compared to related Rhizobiaceae to better understand 'Ca. L. solanacearum' physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, 'Ca. L. solanacearum' is related to 'Ca. L. asiaticus', a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to 'Ca. L. asiaticus', 'Ca. L. solanacearum' probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae.

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Complete genome sequence of Nitrosomonas sp. Is79, an ammonia oxidizing bacterium adapted to low ammonium concentrations

Nitrosomonas sp. Is79 is a chemolithoautotrophic ammonia-oxidizing bacterium that belongs to the family Nitrosomonadaceae within the phylum Proteobacteria. Ammonia oxidation is the first step of nitrification, an important process in the global nitrogen cycle ultimately resulting in the production of nitrate. Nitrosomonas sp. Is79 is an ammonia oxidizer of high interest because it is adapted to low ammonium and can be found in freshwater environments around the world. The 3,783,444-bp chromosome with a total of 3,553 protein coding genes and 44 RNA genes was sequenced by the DOE-Joint Genome Institute Program CSP 2006.     

Members of the Cytophaga–Flavobacterium–Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host ( Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga – Flavobacterium – Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as ' Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of ' Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.     

Using the dog genome to find single nucleotide polymorphisms in red foxes and other distantly related members of the Canidae

Single nucleotide polymorphisms (SNP) are the ideal marker for characterizing genomic variation but can be difficult to find in nonmodel species. We explored the usefulness of the dog genome for finding SNPs in distantly related nonmodel canids and evaluated so-ascertained SNPs. Using 40 primer pairs designed from randomly selected bacterial artificial chromosome clones from the dog genome, we successfully sequenced 80-88% of loci in a coyote (Canis latrans), grey fox (Urocyon cinereoargenteus), and red fox (Vulpes vulpes), which compared favourably to a 60% success rate for each species using 10 primer pairs conserved across mammals. Loci were minimally heterogeneous with respect to SNP density, which was similar, overall, in a discovery panel of nine red foxes to that previously reported for a panel of eight wolves (Canis lupus). Additionally, individual heterozygosity was similar across the three canids in this study. However, the proportion of SNP sites shared with the dog decreased with phylogenetic divergence, with no SNPs shared between red foxes and dogs. Density of interspecific SNPs increased approximately linearly with divergence time between species. Using red foxes from three populations, we estimated F(ST) based on each of 42 SNPs and 14 microsatellites and simulated null distributions conditioned on each marker type. Relative to SNPs, microsatellites systematically underestimated F(ST) and produced biased null distributions, indicating that SNPs are superior markers for these functions. By reconstituting the frequency spectrum of SNPs discovered in nine red foxes, we discovered an estimated 77-89% of all SNPs (within the region screened) present in North American red foxes. In sum, these findings indicate that information from the dog genome enables easy ascertainment of random and gene-linked SNPs throughout the Canidae and illustrate the value of SNPs in ecological and evolutionary genetics.     

Evidence for existence of "mesotogas," members of the order Thermotogales adapted to low-temperature environments

All cultivated isolates of the bacterial order Thermotogales are either thermophiles or hyperthermophiles, but Thermotogales 16S rRNA gene sequences have been detected in many mesophilic anaerobic and microaerophilic environments, particularly within communities involved in the remediation of pollutants. Here we provide metagenomic evidence for the existence of Thermotogales lineages, which we informally call "mesotoga," that are adapted to growth at lower temperatures. Two fosmid clones containing mesotoga DNA, originating from a low-temperature enrichment culture that degrades a polychlorinated biphenyl congener, were sequenced. Phylogenetic analysis clearly puts this bacterial lineage within the Thermotogales order, with the rRNA gene trees and 21 of 58 open reading frames strongly supporting this relationship. An analysis of protein sequence composition showed that mesotoga proteins are adapted to function at lower temperatures than are their identifiable homologs from thermophilic and hyperthermophilic members of the order Thermotogales, supporting the notion that this bacterium lives and grows optimally at lower temperatures. The phylogenetic analysis suggests that the mesotoga lineage from which our fosmids derive has used both the acquisition of genes from its neighbors and the modification of existing thermophilic sequences to adapt to a mesophilic lifestyle.     

Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii

Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole. Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella.     

Comparison of the mesophilic cellulosome‐producing Clostridium cellulovorans genome with other cellulosome‐related clostridial genomes

Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole‐genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901–902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome‐producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate‐degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non‐cellulosomal genes encoding hemicellulases and pectin‐degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome‐producing Clostridium strains for industrial applications such as biofuel production.