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An individual based model of rippling movement in a myxobacteria population.

Migrating cells of Myxococcus xanthus (MX) in the early stages of starvation-induced development exhibit elaborate patterns of propagating waves. These so-called rippling patterns are formed by two sets of waves travelling in opposite directions. It has been experimentally shown that formation of these waves is mediated by cell-cell contact signalling (C-signalling). Here, we develop an individual-based model to study the formation of rippling patterns in MX populations. Following the work of Igoshin et al. (Proc. Natl. Acad. Sci. 98 (2001) 14913) we consider each moving cell to have an internal clock which controls its turning behaviour and sensitivity to C-signal. Specifically, we examine the effects of changing: C-signal strength, sensitivity/refractoriness, cell density, and noise upon the formation and structure of the rippling patterns. We also consider three modified models that have no explicit refractory period and examine their ability to produce rippling patterns.

Communication↗

Evolutionary diversity of ketoacyl synthases in cellulolytic myxobacterium Sorangium.

The diversity of type I polyketide synthases (PKSs) in cellulolytic myxobacterium Sorangium was explored by assaying the ketoacyl synthases (KSs) in 10 Sorangium strains with two degenerate primer sets and 64 different KS fragments were obtained. For their deduced amino acid sequences, eight were identical to three known KSs from Sorangium and Magnetospirillum, while the others showed 54-83% identities to the modular KS domains reported from various microorganisms. Parts of the Sorangium KSs tightly share the clade with Actinobacteria excluding any other analyzed myxobacterial KSs, or with Cyanobacteria /Myxobacteria. Parts are widely located in the three functional groups - "Loading", "NRPS/PKS" and "Trans-AT". Sorangium KSs in the Actinobacteria, Cyanobacteria/Myxobacteria, or "Loading" clade further evolved independently on its own genus. Notably, the modular KSs from other Myxobacteria genera, i.e. Myxococcus, Stigmatella, Melittangium, Cystobacter and Angiococcus are often distributed crosswise and form non-Sorangium blend subgroups. "NRPS/PKS" and "Trans-AT" are two rather diverse groups and the Sorangium KSs in these clades evolved crosswise with other taxa lineages. The results presented in this paper suggest that the inherent genetic strategies, together with frequent gene importing from many organisms (HGT) have contributed to the evolution of modular PKSs in Sorangium. These findings reinforce that Sorangium strains are really excellent creators for novel and diverse polyketides.

Evolution, Molecular↗

Bacteriolytic myxobacteria: parasites or scavengers?

The activity range of an enzymatic preparation from Myxococcus xanthus was assayed against some soil and rhizospheric bacteria. The lysis of unequally aged sensitive eubacterial populations, as related to their viability, was investigated and, at last, the often used streak method was checked and the relationship between cell viability and susceptibility to lysis is discussed.

Bacteriolysis↗

Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii.

In the survey of 14 species of laboratory-cultured cyanobacteria for hemagglutinins, we newly detected the activity in two species, Oscillatoria agardhii, strain NIES-204, and Phormidium foveolarum, strain NIES-503. From the extract of O. agardhii, which showed the highest activity with trypsin-treated erythrocytes of rabbit, a lectin was purified to homogeneity by the combination of precipitation with (NH4)2SO4, gel filtration, hydrophobic chromatography and reverse phase chromatography. The purified lectin, designated OAA, was a monomeric protein with an apparent molecular weight of 13,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 16,000 on gel filtration. The amino acid composition was rich in glycine and acidic amino acids. The hemagglutination activity was inhibited by glycoproteins such as yeast mannan, but not by any of the monosaccharides tested. The activity was stable over a wide range of pH (4-11) and at a high temperature of 80 degrees C, and independent on the presence of divalent cations. The features of OAA resembled those of many of lectins from marine macroalgae. The sequence of amino-terminal residues of OAA was determined as ALYNVENQWGGSSAPWNEGG, which was highly homologous to those of lectins from macroalgae of the genus Eucheuma and that of a myxobacterium Myxococcus xanthus hemagglutinin.

Amino Acid Motifs↗

Retron reverse transcriptase rrtT is ubiquitous in strains of Salmonella enterica serovar Typhimurium.

Bacterial retron reverse transcriptases are unusual enzymes which utilise the same RNA molecule as a template and also as a primer for initiation of the reverse transcription. Except for their relatively frequent presence in Myxococcus spp., they are considered as quite rare proteins. However, in this study we proved that retron reverse transcriptase is frequently found in certain serovars of Salmonella enterica. Using polymerase chain reaction (PCR), in strains of serovar Typhimurium, the rrtT (retron reverse transcriptase Typhimurium) gene was detected in 158 out of 175 tested field strains. On the other hand, in none of the 18 tested serovar Enteritidis strains the rrtT was detected in their genome. Detailed computer analysis allowed us to predict the sequence of msDNA and to propose that the final msDNA is free of any RNA. Furthermore, we predict that there are at least three different classes of retron reverse transcriptases.

Base Sequence↗

Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system--a review.

Genetic analysis of Pseudomonas aeruginosa pilus biogenesis and twitching motility has revealed the requirement for several pil loci which have been localized to different regions of the chromosome. One pil locus, designated pilE, resides at approx. 71 min on the PAO genetic map, a region of the chromosome previously shown to harbor a number of genes required for pilus assembly (i.e., pilA, -B, -C, -D, -R and -S). The PilE protein shows significant sequence identity to the N-terminal domain of PilA as well as to the pilin precursors from a variety of type-4 pilus producers. Included within this homologous region is a short, positively charged leader sequence followed by a prepilin peptidase cleavage site and a largely hydrophobic region. Additionally, an unlinked set of pil genes, designated pilG, -H, -I, -J and -K, has been localized to the SpeI fragment H which corresponds to approx. 20 min on the PAO genetic map. This gene cluster encodes proteins that demonstrate remarkable similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus and are thought to be part of a signal transduction system that controls P. aeruginosa pilus biosynthesis and twitching motility.

Bacterial Outer Membrane Proteins↗

Cloning, sequence analysis and disruption of the mglA gene involved in swarming motility of Sorangium cellulosum So ce26, a producer of the antifungal polyketide antibiotic soraphen A.

The myxobacterium Sorangium cellulosum So ce26, the producer of the agriculturally important fungicide antibiotic soraphen A, displays coordinated gliding motility (swarming) on agar surfaces. The consequent failure to form detached colonies represents a major obstacle for microbiological and genetic studies, since single cells representing discrete genetic events cannot be reliably separated and propagated as clones. The MglA protein, the product of the mglA gene, has been shown to be a central regulator of gliding motility and swarming in the related myxobacterium Myxococcus xanthus. We have cloned and sequenced a chromosomal locus from S. cellulosum So ce26 that shows similarity to the M. xanthus mglA locus. Insertional inactivation of the chromosomal copy of the S. cellulosum So ce26 mglA homolog resulted in a strain with a non-swarming colony phenotype. This strain is able to form distinct colonies presumably derived from single cells. This is the first report on the characterization of a genetic element of the gliding motility system in the myxobacterial suborder Sorangineae.

Journal Article↗

Biosynthesis of iso-fatty acids in myxobacteria: iso-even fatty acids are derived by alpha-oxidation from iso-odd fatty acids.

The biosynthesis of iso-fatty acids was investigated in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca by feeding labeled precursors of these fatty acids and by conducting detailed analysis of the fatty acid profiles and fatty acid MS and NMR spectra. These experiments revealed that in S. aurantiaca all iso-even fatty acids are formed via alpha-oxidation of iso-odd fatty acids and not by using valine-derived isobutyryl-CoA.

Amino Acids↗

Cloning and expression of a Porphyromonas gingivalis gene for protoporphyrinogen oxidase by complementation of a hemG mutant of Escherichia coli.

Porphyromonas gingivalis, a bacterium implicated in periodontal pathogenesis, has a growth requirement for iron protoporphyrin IX. By complementation with a P. gingivalis 381 chromosomal DNA library, we were able to isolate a clone that enhanced the poor growth of a hemG mutant of Escherichia coli. The DNA sequence analysis of this clone revealed three open reading frames (ORFs). ORF3 encoded a protein of 466 amino acids with a calculated molecular weight of 51 695 Da. The deduced amino acid sequence of the ORF3 gene had significant similarity to sequences of protoporphyrinogen oxidase (PPO) from Myxococcus xanthus (30% identical residues). When the ORF3 gene was overexpressed in E. coli, the extract had much higher PPO activity than a control extract, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Thus, ORF3 was named PgHemG. Furthermore, several porphyrin-related genes, including hemD, hemN and hemH, were identified in the data bases on the websites available on-line. We postulated that a porphyrin biosynthetic pathway to heme from preuroporphyrin may be conserved in P. gingivalis.

Amino Acid Sequence↗

A bacterial calcium-binding protein homologous to calmodulin.

Many of the effects of calcium ions in eukaryotic cells are mediated by calcium-binding regulatory proteins such as calmodulin, in which each calcium-binding site has a distinctive helix-loop-helix conformation termed the EF hand. Protein S from the spore coat of the Gram-negative bacterium Myxococcus xanthus has been shown to resemble calmodulin in its internally-duplicated structure and ability to bind calcium. However, it has a beta-sheet secondary structure rather than the helix-loop-helix arrangement of the eukaryotic proteins. We have determined the complete amino-acid sequence of a calcium-binding protein from the Gram-positive bacterium "Streptomyces erythraeus" by cloning and sequencing the corresponding gene. It contains four EF-hand motifs bearing remarkable sequence similarity to the calcium-binding sites in calmodulin. This implies that the EF-hand super-family may have evolved from ancient proteins present in prokaryotes.

Amino Acid Sequence↗

Biosynthesis of iso-fatty acids in myxobacteria.

The fatty acid (FA) profiles of the myxobacteria Stigmatella aurantiaca and Myxococcus xanthus were investigated by acidic methanolysis of total cell extracts and GC or GC-MS analysis. The main components were 13-methyltetradecanoic acid (iso-15:0) and (Z)-hexadec-11-enoic acid (16:1, omega-5 cis). The biosynthesis of iso-FAs was investigated in several feeding experiments. Feeding of isovaleric acid (IVA) to a mutant impaired in the degradation of leucine to isovaleryl-CoA (IV-CoA)(bkd mutant) of M. xanthus only increased the amount of iso-odd FAs, whereas feeding of isobutyric acid (IBA) gave increased amounts only of iso-even FAs. In contrast, a bkd mutant of S. aurantiaca gave increased amounts of iso-odd and iso-even fatty acids in both experiments. We assumed that in S. aurantiacaalpha-oxidation takes place. [D(7)]-15-Methylhexadecanoic acid was synthesised and fed to S. aurantiaca as well as [D(10)]leucine and [D(8)]valine to elucidate this pathway in more detail. The iso-fatty acid was degraded by alpha- and beta-oxidation steps. [D(10)]Leucine was strongly incorporated into iso-odd and iso-even fatty acids, whereas the incorporation rates for [D(8)]valine into both types of fatty acids were low. Thus alpha-oxidation plays an important role in the biosynthesis of iso-fatty acids in S. aurantiaca. The incorporation rates observed after feeding of [D(10)]leucine and [D(8)]valine are the highest for iso-17:0 compared to the other acids. This indicates the central role of iso-17:0 in the biosynthesis of iso-FAs. The shorter homologues seem to be formed mainly by alpha-oxidation and beta-oxidation of this acid. After feeding of traces of unsaturated counterparts of this labelled FA occurred in the extracts indicating that desaturases are active in the biosynthesis of unsaturated fatty acids in S. aurantiaca.

Chromatography, Thin Layer↗

A new class of [2Fe-2S]-cluster-containing protoporphyrin (IX) ferrochelatases.

Protoporphyrin (IX) ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin IX to form haem. These ferrochelatases exist as monomers and dimers, both with and without [2Fe-2S] clusters. The motifs for [2Fe-2S] cluster co-ordination are varied, but in all cases previously reported, three of the four cysteine ligands are present in the 30 C-terminal residues and the fourth ligand is internal. In the present study, we demonstrate that a group of micro-organisms exist which possess protoporphyrin (IX) ferrochelatases containing [2Fe-2S] clusters that are co-ordinated by a group of four cysteine residues contained in an internal amino acid segment of approx. 20 residues in length. This suggests that these ferrochelatases have evolved along a different lineage than other bacterial protoporphyrin (IX) ferrochelatases. For example, Myxococcus xanthus protoporphyrin (IX) ferrochelatase ligates a [2Fe-2S] cluster via cysteine residues present in an internal segment. Site-directed mutagenesis of this ferrochelatase demonstrates that changing one cysteine ligand into serine results in loss of the cluster, but unlike eukaryotic protoporphyrin (IX) ferrochelatases, this enzyme retains its activity. These data support a role for the [2Fe-2S] cluster in iron affinity, and strongly suggest convergent evolution of this feature in prokaryotes.

Amino Acid Motifs↗

The extracytoplasmic function sigma factors: role and regulation.

Alternative sigma factors provide a means of regulating gene expression in response to various extracellular changes. One such class of sigma factors appears to control a variety of functions, including expression of heat-shock genes in Escherichia coli, biosynthesis of alginates and carotenoids in Pseudomonas aeruginosa and Myxococcus xanthus, respectively, iron uptake in E. coli and Pseudomonas spp., nickel and cobalt efflux in Alcaligenes europhus, plant pathogenicity in Pseudomonas syringae and synthesis of outer membrane proteins in Photobacterium sp. strain SS9. Most of these activities deal with extracytoplasmic functions, and such sigmas have been designated as ECF sigma factors. They have also been characterized in Mycobacteria as well as gram-positive bacteria such as Streptomyces coelicolor and Bacillus subtilus and the archaea Sulpholobus acidocaldarius. ECF factors belong to a subfamily of the sigma 70 class, based on their sequence conservation and function across bacterial species. The promoter consensus sequences recognized by the ECF factors are also highly conserved. In most of the cases, the activity of these factors is modulated by a cognate inner membrane protein that has been shown, both in E. coli and in P. aeruginosa, to act as an anti-sigma activity. This inner membrane protein is presumed to serve as a sensor and signalling molecule, allowing an adaptive response to specific environmental change. Presumably, an on-and-off switch of the anti-sigma activity leads to the release of the sigma factor and thereby to the co-ordinate transcription of the specific regulon it governs.

Gene Expression Regulation, Bacterial↗

PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the Gram-negative aetiological agent of gonorrhoeae, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp-dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal PilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tpf. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in PilT. Piliated PilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence-specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT-deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.

Adenosine Triphosphatases↗

Secretion of ATP-utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?

Mycobacterium bovis BCG secretes two ATP-scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9-grown M. bovis BCG cells and determination of the N-terminal amino-acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP-utilizing enzymes was explored. External ATP is important in the activation of macrophage surface-associated P2Z receptors, whose activation has been postulated to allow phagosome-lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP-induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome-lysosome fusion or macrophage apoptotic death.

Adenosine Triphosphatases↗

Alternate routes to conformational specificity in a Greek key beta barrel protein.

The N-terminal domain of protein S, a Greek key calcium-binding protein from Myxococcus xanthus, forms an atypical molten globule in the calcium-free state. The structure of this state is characterized by significant conformational fluctuations, which are localized to a subdomain that is not contiguous along the polypeptide chain. The conformational instability of this subdomain appears to arise from repulsive electrostatic interactions of four acidic side chains that are clustered together but are removed from the calcium-binding sites. This domain can be induced to form a native-like state through two different routes, calcium binding or reduction of pH. Acid-induced folding stabilizes the locally unfolded subdomain by selectively removing repulsive interactions without significantly affecting global stability. In contrast, calcium binding appears to increase local stability indirectly by causing global stabilization.

Calcium↗

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis.

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.

Amino Acid Sequence↗

Inorganic polyphosphate kinase and adenylate kinase participate in the polyphosphate:AMP phosphotransferase activity of Escherichia coli.

Polyphosphate kinase (PPK), responsible for the processive synthesis of inorganic polyphosphate (polyP) from ATP in Escherichia coli, can transfer in reverse the terminal phosphate residue of polyP to ADP to yield ATP. PolyP also serves as a donor in a polyP:AMP phosphotransferase (PAP) activity observed in extracts of Acinetobacter johnsonii and Myxococcus xanthus. We have found that overexpression of the gene encoding PPK results in a large enhancement of PAP activity in E. coli. The PAP activity requires both PPK and adenylate kinase in equimolar amounts. PPK and adenylate kinase form a complex in the presence of polyphosphate. We discuss a phosphotransfer mechanism that involves both enzymes and enables polyP to be a phospho-donor to AMP.

Adenylate Kinase↗