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Robert Haselkorn

Publications and source records attributed to Robert Haselkorn.

17 recordsLinked to original sources

The cyanobacterial genome core and the origin of photosynthesis.

Comparative analysis of 15 complete cyanobacterial genome sequences, including "near minimal" genomes of five strains of Prochlorococcus spp., revealed 1,054 protein families [core cyanobacterial clusters of orthologous groups of proteins (core CyOGs)] encoded in at least 14 of them. The majority of the core CyOGs are involved in central cellular functions that are shared with other bacteria; 50 core CyOGs are specific for cyanobacteria, whereas 84 are exclusively shared by cyanobacteria and plants and/or other plastid-carrying eukaryotes, such as diatoms or apicomplexans. The latter group includes 35 families of uncharacterized proteins, which could also be involved in photosynthesis. Only a few components of cyanobacterial photosynthetic machinery are represented in the genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus, or Heliobacillus mobilis. These observations, coupled with recent geological data on the properties of the ancient phototrophs, suggest that photosynthesis originated in the cyanobacterial lineage under the selective pressures of UV light and depletion of electron donors. We propose that the first phototrophs were anaerobic ancestors of cyanobacteria ("procyanobacteria") that conducted anoxygenic photosynthesis using a photosystem I-like reaction center, somewhat similar to the heterocysts of modern filamentous cyanobacteria. From procyanobacteria, photosynthesis spread to other phyla by way of lateral gene transfer.

Bacterial Proteins↗

Cyanobacterial response regulator PatA contains a conserved N-terminal domain (PATAN) with an alpha-helical insertion.

The cyanobacterium Anabaena (Nostoc) PCC 7120 responds to starvation for nitrogen compounds by differentiating approximately every 10th cell in the filament into nitrogen-fixing cells called heterocysts. Heterocyst formation is subject to complex regulation, which involves an unusual response regulator PatA that contains a CheY-like phosphoacceptor (receiver, REC) domain at its C-terminus. PatA-like response regulators are widespread in cyanobacteria; one of them regulates phototaxis in Synechocystis PCC 6803. Sequence analysis of PatA revealed, in addition to the REC domain, a previously undetected, conserved domain, which we named PATAN (after PatA N-terminus), and a potential helix-turn-helix (HTH) domain. PATAN domains are encoded in a variety of environmental bacteria and archaea, often in several copies per genome, and are typically associated with REC, Roadblock and other signal transduction domains, or with DNA-binding HTH domains. Many PATAN domains contain insertions of a small additional domain, termed alpha-clip, which is predicted to form a four-helix bundle. PATAN domains appear to participate in protein-protein interactions that regulate gliding motility and processes of cell development and differentiation in cyanobacteria and some proteobacteria, such as Myxococcus xanthus and Geobacter sulfurreducens.

Amino Acid Sequence↗

Inactivation of patS and hetN causes lethal levels of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. PCC 7120.

Summary In the filamentous cyanobacterium Anabaena sp. PCC 7120 patS and hetN suppress the differentiation of vegetative cells into nitrogen-fixing heterocysts to establish and maintain a pattern of single heterocysts separated by approximately 10 undifferentiated vegetative cells. Here we show that the patS- and hetN-dependent suppression pathways are the only major factors that prevent vegetative cells from differentiating into heterocysts when a source of ammonia is not present. The patS and hetN pathways are independent of each other, and inactivation of both patS and hetN leads to differentiation of almost all cells of a filament in the absence of a source of fixed nitrogen, compared with approximately 9% in the wild type. Complete differentiation of filaments also occurs when nitrate is supplied as a source of fixed nitrogen, conditions that do not induce differentiation of wild-type filaments. However, ammonia is still capable of suppressing differentiation. The percentage of cells that differentiate into heterocysts appears to be a function of time when a source of fixed nitrogen is absent or a function of growth phase when nitrate is supplied. Although differentiation proceeds unchecked in the absence of patS and hetN expression, differentiation is asynchronous and non-random.

Actin Cytoskeleton↗

Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90.

The cluster of microcystin synthetase genes from Anabaena strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (mcyA-mcyB-mcyC) is transcribed in the opposite direction from the second operon (mcyG-mcyD-mcyJ-mcyE-mcyF-mcyI) and the third operon (mcyH). The genes mcyA, mcyB, and mcyC encode nonribosomal peptide synthetases (NRPS), while mcyD codes for a polyketide synthase (PKS), and mcyG and mcyE are mixed NRPS-PKS genes. The genes mcyJ, mcyF, and mcyI are similar to genes coding for a methyltransferase, an aspartate racemase, and a D-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of mcyB coding for the adenylation domain was found to be 96% identical with the corresponding part of mcyC, suggesting a recent duplication of this fragment and a replacement in mcyB. In Anabaena strain 90, the order of the domains encoded by the genes in the two sets (from mcyG to mcyI and from mcyA to mcyC) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in Anabaena strain 90 differs from the arrangement found in two other cyanobacterial species, Microcystis aeruginosa and Planktothrix agardhii. The average sequence match between the microcystin synthetase genes of Anabaena strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.

Amino Acid Sequence↗

Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis.

Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndromes. It is closely related to the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the former being used as a biological weapon and the latter as a pesticide. B. anthracis and B. thuringiensis are readily distinguished from B. cereus by the presence of plasmid-borne specific toxins (B. anthracis and B. thuringiensis) and capsule (B. anthracis). But phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus, B. anthracis and B. thuringiensis are varieties of the same species or different species. Here we report the sequencing and analysis of the type strain B. cereus ATCC 14579. The complete genome sequence of B. cereus ATCC 14579 together with the gapped genome of B. anthracis A2012 enables us to perform comparative analysis, and hence to identify the genes that are conserved between B. cereus and B. anthracis, and the genes that are unique for each species. We use the former to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers.

Bacillus anthracis↗

Genome analysis of F. nucleatum sub spp vincentii and its comparison with the genome of F. nucleatum ATCC 25586.

We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp. vincentii (FNV), and compare that genome with F. nucleatum ATCC 25586 (FN). A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified. Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms. In addition to the excretion of butyrate, H2S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate. Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen. It appears to transport ferrous iron by an anaerobic ferrous transporter. Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV. ORFs for ethanolamine utilization, thermostable carboxypeptidase, gamma glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV. FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present. These capabilities allow Fusobacteria to survive in a mixed culture in the mouth.

ATP-Binding Cassette Transporters↗

Expression of cytosolic and plastid acetyl-coenzyme A carboxylase genes in young wheat plants.

Expression of cytosolic and plastid acetyl-coenzyme A carboxylase (ACCase) gene families at the mRNA level was analyzed in developing wheat (Triticum aestivum) plants. The major plastid ACCase mRNA level is high in the middle part of the plant and low in roots and leaf blades. An alternative plastid ACCase transcript initiated at a different promoter and using an alternative 5' splice site for the first intron accumulates to its highest level in roots. Cytosolic ACCase mRNA also consists of two species, one of which is present at approximately a constant level, whereas the other accumulates to a high level in the lower sheath section. It is likely that different promoters are also responsible for the two forms of cytosolic ACCase mRNA. The abundances of cytosolic and plastid ACCase mRNAs in the sheath section of the plant are similar. ACCase protein level is significantly lower in the leaf blades, in parallel with changes in the total ACCase mRNA level. Homoeologous ACCase genes show the same expression patterns and similar mRNA levels, suggesting that none of the genes was silenced or acquired new tissue specificity after polyploidization.

Acetyl-CoA Carboxylase↗

Heterocyst-specific expression of patB, a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120.

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.

Anabaena↗

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains.

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two "gapped" Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an "in silico" cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

Bacterial Proteins↗

Microarray analysis of gene expression during bacteriophage T4 infection.

Genomic microarrays were used to examine the complex temporal program of gene expression exhibited by bacteriophage T4 during the course of development. The microarray data confirm the existence of distinct early, middle, and late transcriptional classes during the bacteriophage replicative cycle. This approach allows assignment of previously uncharacterized genes to specific temporal classes. The genomic expression data verify many promoter assignments and predict the existence of previously unidentified promoters.

Bacteriophage T4↗

Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex and the evolutionary history of polyploid wheat.

The classic wheat evolutionary history is one of adaptive radiation of the diploid Triticum/Aegilops species (A, S, D), genome convergence and divergence of the tetraploid (Triticum turgidum AABB, and Triticum timopheevii AAGG) and hexaploid (Triticum aestivum, AABBDD) species. We analyzed Acc-1 (plastid acetyl-CoA carboxylase) and Pgk-1 (plastid 3-phosphoglycerate kinase) genes to determine phylogenetic relationships among Triticum and Aegilops species of the wheat lineage and to establish the timeline of wheat evolution based on gene sequence comparisons. Triticum urartu was confirmed as the A genome donor of tetraploid and hexaploid wheat. The A genome of polyploid wheat diverged from T. urartu less than half a million years ago (MYA), indicating a relatively recent origin of polyploid wheat. The D genome sequences of T. aestivum and Aegilops tauschii are identical, confirming that T. aestivum arose from hybridization of T. turgidum and Ae. tauschii only 8,000 years ago. The diploid Triticum and Aegilops progenitors of the A, B, D, G, and S genomes all radiated 2.5-4.5 MYA. Our data suggest that the Acc-1 and Pgk-1 loci have different histories in different lineages, indicating genome mosaicity and significant intraspecific differentiation. Some loci of the S genome of Aegilops speltoides and the G genome of T. timophevii are closely related, suggesting the same origin of some parts of their genomes. None of the Aegilops genomes analyzed is a close relative of the B genome, so the diploid progenitor of the B genome remains unknown.

Acetyl-CoA Carboxylase↗

The carboxyltransferase activity of the apicoplast acetyl-CoA carboxylase of Toxoplasma gondii is the target of aryloxyphenoxypropionate inhibitors.

Inhibition of growth of the apicomplexan parasite Toxoplasma gondii by aryloxyphenoxypropionate herbicides has been correlated with the inhibition of its acetyl-CoA carboxylase (ACC) by these compounds. Here, full-length and C-terminal fragments of T. gondii apicoplast ACC as well as C-terminal fragments of the cytosolic ACC were expressed in Escherichia coli. The recombinant proteins that were soluble showed the expected enzymatic activities. Yeast gene-replacement strains depending for growth on the expressed T. gondii ACC were derived by complementation of a yeast ACC1 null mutation. In vitro and in vivo tests with aryloxyphenoxypropionates showed that the carboxyltransferase domain of the apicoplast T. gondii ACC is the target for this class of inhibitors. The cytosolic T. gondii ACC is resistant to aryloxyphenoxypropionates. Both T. gondii isozymes are resistant to cyclohexanediones, another class of inhibitors targeting the ACC of grass plastids.

Acetyl-CoA Carboxylase↗

Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights.

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

Attachment Sites, Microbiological↗

Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586.

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.

Amino Acids↗

Newly identified cytochrome c oxidase operon in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 specifically induced in heterocysts.

Two operons have been cloned from Anabaena sp. strain PCC 7120 DNA, each of which encodes the three core subunits of distinct mitochondrial-type cytochrome c oxidases. The two operons are only 72 to 85% similar to one another at the nucleotide level in the most conserved subunit. One of these, coxBACII, is induced >20-fold in the middle to late stages of heterocyst differentiation. Analysis of green fluorescent protein reporters indicates that this operon is expressed specifically in proheterocysts and heterocysts. The other operon, coxBACI, is induced only 2.5-fold following nitrogen step-down and is expressed in all cells. Surprisingly, a disruption mutant of coxAII, the gene encoding subunit I of the heterocyst-specific oxidase, grows normally in the absence of combined nitrogen. It is likely that coxBACI and/or two other putative terminal oxidases present in the Anabaena sp. strain PCC 7120 genome are able to compensate for the loss of the heterocyst-specific oxidase in providing ATP for nitrogen fixation and maintaining a low oxygen level in heterocysts.

Anabaena↗

The genome sequence of the facultative intracellular pathogen Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

Brucella melitensis↗

Phylogenetic analysis of the acetyl-CoA carboxylase and 3-phosphoglycerate kinase loci in wheat and other grasses.

We have applied a two-gene system based on the sequences of nuclear genes encoding multi-domain plastid acetyl-CoA carboxylase (ACCase) and plastid 3-phosphoglycerate kinase (PGK) to study grass evolution. Our analysis revealed that these genes are single-copy in most of the grass species studied, allowing the establishment of orthologous relationships between them. These relationships are consistent with the known facts of their evolution: the eukaryotic origin of the plastid ACCase, created by duplication of a gene encoding the cytosolic multi-domain ACCase gene early in grass evolution, and the prokaryotic (endosymbiont) origin of the plastid PGK. The major phylogenetic relationships among grasses deduced from the nucleotide sequence comparisons of ACCase and PGK genes are consistent with each other and with the milestones of grass evolution revealed by other methods. Nucleotide substitution rates were calculated based on multiple pairwise sequence comparisons. On a relative basis, with the divergence of the Pooideae and Panicoideae subfamilies set at 60 million years ago (MYA), events leading to the Triticum/Aegilops complex occurred at the following intervals: divergence of Lolium (Lolium rigidum) at 35 MYA, divergence of Hordeum (Hordeum vulgare) at 11 MYA and divergence of Secale (Secale cereale) at 7 MYA. On the same scale, gene duplication leading to the multi-domain plastid ACCase in grasses occurred at 129 MYA, divergence of grass and dicot plastid PGK genes at 137 MYA, and divergence of grass and dicot cytosolic PGK genes at 155 MYA. The ACCase and PGK genes provide a well-understood two-locus system to study grass phylogeny, evolution and systematics.

Acetyl-CoA Carboxylase↗