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Two-component systems (TCSs) are a large family of signalling pathways characterized by the successive transfer of phosphoryl groups between the histidine and aspartate residues of paired histidine kinase and response regulator proteins. With the availability of genome sequences for four genera of myxobacteria it has become possible to assess the genomic complements of myxobacterial TCS genes and to characterize features of their organization and evolutionary heritage. In this study we have compiled lists of the TCS genes within myxobacterial genomes and characterized their domain architecture, gene organization and evolutionary relationships. In order to provide an appropriate context for our conclusions, where possible we have compared myxobacterial TCSs with those found in 316 other completely sequenced bacteria. Myxobacteria have the largest number of TCSs of any organisms. An unusually low proportion of TCS genes are paired in myxobacterial genomes, and myxobacterial histidine kinases also seem to sense internal signals to an unusual degree. Phylogenetic evidence has allowed us to suggest homologous relationships of proteins across the myxobacteria, and it appears that myxobacterial TCS evolution has been dominated by duplications, gene rearrangements and changes in sensory domain complements. The systematic classification of the TCS proteins of the myxobacteria presented here should also provide a framework for future experimental studies on two-component regulation in these organisms.

Aims: Efficient strain dereplication is of great value during the generation of bacterial strain collections for industrial screening. We evaluated the utilization of the RNase P RNA gene (rnpB) sequence as a tool for molecular dereplication of myxobacteria., Methods and Results: 16S rDNA (approx. 1 x 5 kbp) and rnpB (approx. 0 x 3 kbp) sequences were obtained and aligned. From 50 strains, we obtained 20 different sequences for the 16S rDNA and 24 for rnpB. Intersequence similarity was lower for rnpB than for 16S rDNA., Conclusions: rnpB allows the rapid discrimination of similar strains, with a higher resolution power as compared with 16S rRNA gene sequencing. It not only gives better discrimination, but is also faster and cheaper than 16S rDNA sequencing., Significance and Impact of the Study: Myxobacteria isolation and cultivation require time and experience. The application of rnpB sequencing to early myxobacterial strain dereplication may help in the generation of diverse strain libraries of these bacteria.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

The diversity of myxobacteria present in campus garden soil was surveyed by both cultivation-based and cultivation-independent methods. Detailed phylogenetic analysis of cultured and uncultured myxobacteria 16S rRNA gene sequences revealed that many undescribed relatives of the myxobacteria exist in nature. Molecular systematic analyses also revealed that myxobacterial genera described to date on the basis of the morphology of multi-cellular fruiting bodies were mostly monophyletic. However, these known taxa comprised only in a small part of the sequences recovered directly from soil in a cultivation-independent approach, indicating that the group is much more diverse than previously thought. We propose that the myxobacteria exist in two forms: the fruiting and the non-fruiting types. Most of the uncultured myxobacteria may represent taxa which rarely form fruiting bodies, or may lack some or all of the developmental genes needed for fruiting body formation. In order to identify non-fruiting myxobacteria, new morphology-independent cultivation and isolation techniques need to be developed.

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.

Sorangium cellulosum strains produce approximately 50% of the biologically active secondary metabolites known from myxobacteria. These metabolites include several compounds of biotechnological importance such as the epothilones and chivosazols, which, respectively, stabilize the tubulin and actin skeletons of eukaryotic cells. S. cellulosum is characterized by its slow growth rate, and natural products are typically produced in low yield. In this study, biomagnetic bead separation of promoter-binding proteins and subsequent inactivation experiments were employed to identify the chivosazol regulator, ChiR, as a positive regulator of chivosazol biosynthesis in the genome-sequenced strain So ce56. Overexpression of chiR under the control of T7A1 promoter in a merodiploid mutant resulted in fivefold overproduction of chivosazol in a kinetic shake flask experiment, and 2.5-fold overproduction by fermentation. Using quantitative reverse transcription PCR and gel shift experiments employing heterologously expressed ChiR, we have shown that transcription of the chivosazol biosynthetic genes (chiA-chiF) is directly controlled by this protein. In addition, we have demonstrated that ChiR serves as a pleiotropic regulator in S. cellulosum, because mutant strains lack the ability to develop into regular fruiting bodies.

Corallococcus coralloides DSM 2259(T), Corallococcus exiguus DSM 14696(T), Corallococcus macrosporus DSM 14697(T) and more than 35 strains identified as members of Corallococcus on the basis of morphology were subjected to partial sequences analysis of three housekeeping genes (lepA, fusA and rpoB), complementing a recent phylogenetic analysis based on genes coding for 16S rRNA and gyrB. Phylogenetic analysis of each gene, generated by maximum likelihood and two different additive treeing algorithms, resulted in the separate position of C. macrosporus DSM 14697(T) and a few Corallococcus strains that were more closely related to Myxococcus xanthus than to the other members of Corallococcus. The latter strains formed three clearly separate clusters by 16S rRNA gene phylogeny. This relationship, however, was only partially recovered by the other gene trees. Group 1, embracing the type strains of C. coralloides and C. exiguus, only emerged as a coherent cluster in the 16S rRNA gene tree. In all other gene trees this cluster embraced organisms of cluster 3, which either formed coherent subclusters (gyrB, lepA) or which appeared polyphyletic (fusA, rpoB). Group 2 organisms consistently constituted a monophyletic cluster, though their branching within the gene trees differed. A concatenated tree, based on the analysis of about 5400 nucleotides of all five partial genes was most similar to the 16S rRNA gene tree. In order to determine whether the individual clusters that emerged by 16S rRNA gene analysis (>99.1% intracluster similarities) show phenetic properties that would allow their description as new species, a few strains of each group were subjected to the analysis of whole cell fatty acid and physiological properties. Riboprint patterns were generated for some members of group 1. While the DNA-DNA reassociation values and riboprint patterns confirmed the genomic heterogeneity of members of cluster 1, none of the other properties investigated were sufficiently discriminative to allow the formal description of strain clusters as new species.

Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH(4) and/or CO(2). Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with (13)C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25 degrees C for 12 days, during which (13)C-labeled acetate was added at a concentration of 500 muM every 3 days. (13)C-labeled CH(4) and CO(2) were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate (13)C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO(2) was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.

A rare, cellulose-decomposing myxobacterium is described, and a new genus name, Byssovorax, is proposed for it. The organism is almost certainly identical to the species 'Myxococcus cruentus' Thaxter 1897, and that species epithet is therefore revived for the novel bacterium: the type strain of Byssovorax cruenta gen. nov., sp. nov., nom. rev. is strain By c2(T) (=DSM 14553(T)=CIP 108850(T)). The G+C content of its DNA is 69.9 mol%. The 16S rRNA gene sequence shows that the species belongs to the family Polyangiaceae, suborder 'Sorangineae', of the Myxococcales. An emended description of the organism is given.

A slightly halophilic myxobacterial strain, SMH-27-4, was isolated from nearshore soil and shown to belong to a new myxobacterium genus based on phylogenetic analysis. This slowly-growing myxobacterium produced the novel antibiotic depsipeptides miuraenamides A and B. Their physico-chemical properties and molecular formulas, C34H42N3O7Br and C34H42N3O7I, were determined. Miuraenamides A exhibited potent and selective inhibition against a phytopathogenic microorganism, Phytophthora sp., and moderate inhibition against some fungi and yeasts, but was ineffective against bacteria. Both of the metabolites inhibited NADH oxidase at IC50 values of 50 microM, suggesting, like beta-methoxyacrylate-type antibiotics, the electron transfer system of the mitochondrial respiratory chain as the cellular target.

Six red-pigmented strains of the Alphaproteobacteria with optimal growth between 45 and 54 degrees C were previously isolated from coloured biofilms in two fine-paper machines and one pulp dryer. The strains were found to be resistant to 15 p.p.m. 2,2-dibromo-3-nitrilopropionamide, a common industrial biocide. 16S RNA gene sequence similarity of the isolates was 99.7-100 %. Ribotyping using the restriction enzymes PvuII and EcoRI showed that four of the isolates (C-lvk-R2A-1, C-lvk-R2A-2(T), C-R2A-52d and C-R2A-5d) belong to a single species. 16S rRNA gene-based phylogenetic analysis revealed that, together with Rhodobacter blasticus ATCC 33485(T), the isolates form a deep line of descent (94.7-94.9 % sequence similarity) within the family Rhodobacteraceae loosely affiliated with the Rhodobacter/Paracoccus clade. The isolates were strictly aerobic and oxidase-positive (catalase was weakly positive) and utilized a wide range of substrates including pentoses, hexoses, oligosaccharides and sugar alcohols. The predominant constituents in their cellular fatty acid profiles were C(19 : 0) cyclo omega8c (39-44 %), C(18 : 0) (21-24 %) and C(16 : 0) (21-23 %). Fatty acids present in smaller amounts included C(18 : 1)omega7c, C(10 : 0) 3-OH, C(18 : 1)omega7c 11-methyl, C(20 : 2)omega6,9c and C(17 : 0) cyclo, amongst others. Polar lipids included diphosphatidylglycerol, phosphatidylcholine and an unidentified aminolipid, but not phosphatidylethanolamine. Carotenoid pigments were synthesized but bacteriochlorophyll a was not. The polyamine patterns consisted of the major compounds putrescine, spermidine and sym-homospermidine. The major respiratory lipoquinone was ubiquinone Q-10. The DNA G+C content was 69.4-70.2 mol%. On the basis of the phylogenetic and phenotypic evidence, the biofilm isolates were classified in a new genus, Rubellimicrobium gen. nov.; four of the isolates are assigned to the type species, Rubellimicrobium thermophilum gen. nov., sp. nov. Strain C-lvk-R2A-2(T) (=CCUG 51817(T) = DSM 16684(T) = HAMBI 2421(T)) is the type strain of Rubellimicrobium thermophilum.

The diversity of myxobacteria in a soil niche was explored using culture-dependent and -independent methods. Conventional cultivation for bacteriolytic myxobacteria produced six types of myxobacteria, which were identified as two Myxococcus spp., two Corallococcus spp., a Cystobacter sp. and a Nannocysts sp. Hybridization analysis of the soil bacterial 16S rRNA gene library with myxobacteria-specific probes revealed that myxobacteria accounted for less than 1% in the bacterial community. A Cystobacterineae 16S rRNA genes-rich library was further established from the soil DNA by polymerase chain reaction amplification with a Cystobacterineae-specific primer combined with a universal bacterial primer. Screening of the special library using Cystobacterineae- and Sorangineae-specific probes produced approximately 45% and 3% positive signals respectively. Sixty-four positive clones were randomly selected for sequencing. Except three repeats, the sequences were diverse ranging from 0.3% to 21.3%, and homologous with the known myxobacteria at 77.6-99.8%, including 57 in Cystobacterineae, one close to Nannocystis and three much more distant from the known myxobacteria. The sequences in the Cystobacterineae can further be divided into at least 12 groups, of which most were unreported. The results suggest that myxobacteria in nature are much more diverse than were ever known, even in one soil niche.

Thirty exopolysaccharides (EPS) produced by bacteria isolated from biofilms or slimelayers from different paper and board mills in Finland, France and Spain were subjected to size exclusion chromatography and sugar compositional analysis. High performance size exclusion chromatography (HPSEC) analysis revealed that some samples were composed of several molecular weight populations. These samples were fractionated by size exclusion chromatography and pooled accordingly. Principal components analysis (PCA) of the sugar compositions of the different pools indicated the presence of glucans and mannans caused by insufficient removal of the carbon or nitrogen source (yeast extract) from the bacteria growth medium leading to an overestimation of the glucose and mannose level in the sample, respectively. From the point of view of slime problems the EPS populations are the most important for multivariate analysis. Four groups of EPSs have been recognized by PCA analysis: a group of EPSs produced by Enterobacter and related genera similar to the regularly reported colanic acid; a group of Methylobacterium EPSs having high galactose and pyruvate levels and two groups that showed less dense clusters produced by Bacillus and related genera, showing high mannose and/or glucose levels and Klebsiella EPSs that showed galactose with rhamnose as major characteristic sugar moieties. Fourier transform infrared spectroscopy (FT-IR) of the same samples followed by discriminant partial least squares regression (DPLS) and linear discriminant analysis (LDA) showed that, when used with a well-defined training set, FT-IR could be used clustering instead of time-consuming sugar composition analysis. The Enterobacter and Methylobacetrium EPS groups could be recognized clearly. However the fact that this could hardly be done for the other two groups in the dataset indicates the importance of a larger and well-defined training or calibration set. The potential to use FT-IR, as a tool for pattern recognition and clustering with respect to EPS structures produced by micro organisms isolated from a paper mill environment is discussed.

Thirty-three strains classified as Corallococcus coralloides isolated from mostly soil samples in 14 countries of four continents, were subjected to phylogenetic analyses. Based on 16S rDNA analyses the strains form a highly related cluster, sharing above 98.7% sequence similarity. Four groups were recognized within this cluster, only one of which, containing two strains from St. Lucia, Lower Antilles, was exclusively defined by strains from the same sample. The other groups contained members from different countries, even continents. The largest group embraced the type strains of C. coralloides DSM 2259(T) and Corallococcus exiguus 14696(T) which were almost indistinguishable in their 16S rRNA gene sequence. Corallococcus macrosporus DSM 14697(T) grouped outside the C. coralloides cluster, showing a higher relationship to a member of Myxococcus. The topology of the tree generated on the basis of the partial gyrase B (gyrB) gene sequence supports the rRNA gene tree, though some differences in the order of branching were observed. As judged by the binary similarity values the higher resolution power of gyrB sequences was confirmed. From a taxonomic standpoint, the size of myxospores is not a valuable taxonomic criterion, as small- and medium-sized myxospores are members of the same group. If the species status of C. coralloides and C. exiguus is verified by other methods (e.g. DNA-DNA hybridisation, RiboTyping), the genus Corallococcus may embrace a broad range of yet-to-be described novel species. The presence of strains within the same sample displaying higher relatedness to strains from other locations points towards an intensive dispersal of myxospores across continents.

Novel moderately thermophilic Myxobacteria from both known suborders (Cystobacterineae and Sorangineae) were isolated from soil samples of semiarid and warm climates. The addition of the anthelmintic and amoebizidal agent levamisole was a new and crucial improvement for the fast isolation of overall 37 strains, which grew very fast at temperatures of 42-44 degrees C. When the 16S rDNAs were compared with GenBank data of common Myxobacteria, identities were 98-99%, thus not reflecting the physiological differences. Similar to the Myxobacteria described so far, the new isolates are multiresistant against a variety of antibiotics and are producers of typical myxobacterial secondary metabolites. Analysis of our previous strain collection isolated from soil samples taken worldwide revealed a more or less uniform distribution of strains which synthesize specific metabolites. Therefore these moderately thermophilic Myxobacteria, which grow 2-3 times faster, have the potential to replace the slow-growing isolates and provide a means for fast and cost-saving production of myxobacterial metabolites in the future.

Nine Corallococcus isolates and three type strains of Corallococcus species were characterized by Intact Cell Mass Spectrometry using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The resulting phenetic clustering was compared to the phylogenetic grouping based upon sequences of two housekeeping genes. The three dendrograms of relatedness resembled each other in that the isolates were highly similar to the type strains of Corallococcus exiguus and Corallococcus coralloides, while Corallococcus macrosporus and Myxococcus xanthus were more distantly related. While certain pairs of organisms were recovered by spectrometry and genes sequence analysis, others were detected by two of the three approaches. The degree of similarity determined by sequence analysis of the two genes was not higher than that revealed by MALDI-TOF analysis. The results show that the spectral profile, consisting of about 25 to 45 masses ranging between 2 and 20 kDa, have indeed taxonomic significance, confirming literature data that ribosomal proteins and certain housekeeping proteins are responsible for the masses obtained. Provided the availability of a database of type strains, MALDI-TOF analysis of unknown strains appears to be a rapid and inexpensive method to taxonomically cluster environmental isolates, expanding the spectrum to strains other than those of medical importance predominantly investigated so far.

The purpose of this study was to establish the myxobacterial biodiversity of an established oak-hickory forest and a savanna restoration site that has been cut and subsequently burned on four occasions between 1993 and 1998 in an attempt to restore the land to the native savanna ecosystem. Soil and bark samples were processed through standard methods specifically for myxobacteria and numbers and types of species were recorded for both locations. Species were identified through morphology of fruiting bodies, SDS-PAGE of whole cell protein profiles, and 16S rRNA gene sequences. Statistical analyses were employed and suggested that significantly greater numbers and types of myxobacteria are present on the bark of the trees in the established oak-hickory forest than the bark of trees in the savanna restoration site, while little difference in numbers and types of species were observed between the soil samples of the two locations.

Aims: To evaluate the patterns of the production of antimicrobial compounds by Israeli myxobacteria newly isolated from soil samples and barks by a battery of isolation and purification methods., Methods and Results: A total of 100 myxobacteria belonging to five of the 12 described genera, were isolated from 48 soil and 45 tree bark samples collected in different areas inside the State of Israel. Four isolation methods based on the peculiar metabolic and cell cycle aspects of myxobacteria, were combined with purification procedures and optimization of cultivation conditions. Ninety-seven strains were fermented and screened for antimicrobial activities. Production of antimicrobial activities was detected in 62 isolates. More than 50% of the collection (54 strains) was able to inhibit Escherichia coli growth., Conclusions: The results of this study support the idea that myxobacterial strains can be isolated from particular habitats and then cultivated and screened for their capacity to produce secondary metabolites endowed with antibacterial and antifungal activities. Myxovirescin, a typical poliketide myxobacterial antibiotic, has been identified in one Israeli isolate. Althiomycin, a thiazolyl peptide, which inhibits prokaryotic protein synthesis, usually produced by actinomycetes, was detected in three strains selected in this study., Significance and Impact of the Study: The results confirm that myxobacteria are prolific producers of a variety of bioactive secondary metabolites including antibacterial and antifungal compounds, being their high frequency of anti-Gram-negative activities particularly appealing for the current anti-infective research. So far their screening has often been hampered because their isolation is time-consuming and are quite difficult to handle and cultivate. In this paper we demonstrate that a proper combination of isolation, purification and cultivation methods allow their pharmaceutical exploitation.

By using baiting techniques and different purification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced to sterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of some myxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study.

The diversity of 16S-23S rDNA intergenic spacer regions (ISR) among cellulolytic myxobacterial strains was assayed. Agarose gel electrophoresis of PCR amplification products from ten strains shows that there are at least four copies of rRNA operons in the genus Sorangium, based on their size and restriction enzymatic digest maps. There are two sequence organization patterns: tRNA(Ile)-tRNA(Ala)-containing ISR and tRNA-lacking ISR. The tRNA-containing ISRs are highly similar among strains and within a strain (more than 98% similarity) and contain the essential functional regions, such as a ribonuclease III recognition site and an antiterminator recognition site boxA. The tRNA-lacking ISR has no such functional sites that are important for yielding mature rRNA, which suggests that this type of rRNA operons might be degenerate. The tRNA-lacking ISR is divided into two types based on their sizes and sequences, which exhibits about 90% similarity within each type. Thus, the tRNA-lacking ISR polymorphisms can be used to discriminate among different strains of sorangial species.

Six isolates of novel marine myxobacteria, designated strains SHK-1T, SMK-1-1, SMK-1-3, SMK-10, SKK-2, and SMP-6, were obtained from various coastal samples (mud, sands and algae) collected around Japan. All of the isolates had Gram-negative rod-shaped cells, motile by gliding and grew aerobically. They showed bacteriolytic action, fruiting body formation, and NaCl requirement for growth with an optimum concentration of 1.0-2.0% (w/v). In addition, divalent cationic components of seawater, such as Mg2+ or Ca2+, were also needed for growth. The major respiratory quinone was MK-7. The G+C content of genomic DNA ranged from 65.6 to 67.4 mol% (by HPLC). The isolates shared almost identical 16S rDNA sequences, and clustered with a recently described marine myxobacterium, Plesiocystis pacifica, as their closest relative on a phylogenetic tree (95.9-96.0% similarity). Physiological and chemotaxonomic differences between the new strains and strains of the genus Plesiocystis justify the proposal of a new genus. Therefore, we propose to classify the six isolates into a new taxon of marine myxobacteria with the name, Enhygromyxa salina gen. nov., sp. nov. The type strain is SHK-1(T) (JCM 11769(T) = DSM 15217(T) = AJ 110011(T)).

The evolutionary distances of the 16S rDNA sequences in cellulolytic myxobacteria are less than 3%, which units all the strains into a single genus, Sorangium. The size of myxospores and the shape of sporangioles, rather than fruiting body colors or swarm morphologies are consistent with the changes of the 16S rDNA sequences. It is suggested that there are at least two species in the genus Sorangium: one includes strains with small myxospores and spherical sporangioles, and the color of the fruiting bodies is normally orange or brown, though sometimes yellow or black. The second species has large myxospores, polyhedral sporangioles with many inter-cystic substrates, and normally deep brown to black color.

Two strains of a novel myxobacterium (designated SIR-1T and SHI-1) were isolated from Japanese coasts located in the Pacific subtropical zone. Cells of both strains were Gram-negative, rod-shaped and motile by gliding. The strains were chemoheterotrophic and strictly aerobic. They had the common characteristics associated with myxobacteria, such as bacteriolytic action and fruiting-body formation. The characteristic feature of the strains was a NaCl growth requirement with an optimum concentration of 2.0-3.0 % (w/v), comparable to that of sea water. In addition, other cationic components of sea water, such as Mg2+, Ca2+ and K+, were needed for growth. The major respiratory quinone was MK-8(H2). The cellular fatty acid profile was characterized by the predominance of iso-C15:0. Characteristic fatty acids anteiso-C16:0 and anteiso-C17:0, and a long-chain polyunsaturated fatty acid (C20:4), were also detected. The G + C content of the genomic DNA of strains SIR-1T and SHI-1 was between 69.3 and 70.0 mol% (as determined by HPLC). Strains SIR-1T and SHI-1 shared almost identical 16S rDNA sequences, and clustered with the genus Nannocystis as their closest relative upon phylogenetic analysis. However, the phylogenetic distance between the novel strains and the genus Nannocystis was large enough to warrant their different generic allocation. This finding was supported by significant phenotypic differences between the novel strains and Nannocystis. Thus, strains SIR-1T and SHI-1 represent a novel genus and species, for which the names Plesiocystis and Plesiocystis pacifica, respectively, are proposed. The type strain of the species is SIR-1T (=JCM 11591T =DSM 14875T =AJ 13960T).

In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain So ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16S rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCC 25531 and the epothilone producer S. cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2 Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.

The marine myxobacterium strain NU-2, which can grow on high concentrations (up to 7%) of NaCl, was isolated from a salt soil sample collected from the coast of the Huanghai Sea, China. Morphological properties and 16S rDNA sequence analysis indicated that the isolate is a novel species related to the genus Nannocystis. Nannocystis sp. NU-2 produced a new kind of flocculating substance in a starch medium with a yield of 14.8 g l(-1). The NU-2 flocculant was composed of 40.3% proteins and 56.5% polysaccharides, of which glucose, mannose and glucuronic acid were the principal constituents in the relative proportions of 5:4:1. The flocculation activity of the NU-2 flocculant depends strongly on cations such as Fe(3+) and Al(3+). When a 30 mg l(-1) FeCl(3) solution is present in kaolin clay suspension, 30 mg l(-1)of the flocculant produced a high flocculating activity value of 90%, which remained unchanged over an extensive pH range (pH 2.0-13.0). The flocculant was tested for its ability to bleach dyeing liquors, and the bleaching activities were 98.2% for acid red in 100 mg l(-1)of the flocculant and 99.0% for direct emerald blue in 50 mg l(-1)of the flocculant under test conditions. Use of the flocculant to bleach basic pink and cation emerald blue liquors was not effective.

Phenotypic and phylogenetic studies were performed on two myxobacterial strains, SMP-2 and SMP-10, isolated from coastal regions. The two strains are morphologically similar, in that both produce yellow fruiting bodies, comprising several sessile sporangioles in dense packs. They are differentiated from known terrestrial myxobacteria on the basis of salt requirements (2-3% NaCl) and the presence of anteiso-branched fatty acids. Comparative 16S rRNA gene sequencing studies revealed that SMP-2 and SMP-10 are genetically related, and constitute a new cluster within the myxobacteria group, together with the Polyangium vitellinum Pl vt1 strain as the closest neighbor. The sequence similarity between the two strains is 95.6%. Based on phenotypic and phylogenetic evidence, it is proposed that these two strains be assigned to a new genus, Haliangium gen. nov., with SMP-2 designated as Haliangium ochraceum sp. nov. (= JCM 11303(T) = DSM 14365(T)), and SMP-10 as Haliangium tepidum sp. nov. (= JCM 11304(T)= DSM 14436(T)).

This paper reported that the authors isolated large numbers of myxobacteria from more than 100 samples collected in many places of China. More than 400 pure strains were obtained. These strains belonged to 10 genera of Myxococcales. The most frequent genera isolated were Myxococcus, Sorangium, Corallococcus, and Cystobacter. Melittangium was seldom isolated. No Chondromyces and Haploangium were found.

This review presents an overview of the present status of the biology of the myxobacteria, including the molecular biology of the systems that control and regulate myxobacterial gliding movement and morphogenesis. The present status of myxobacterial taxonomy and phylogeny is described. The evolutionary biology of the myxobacteria is emphasized with respect to their social behavior and the molecular basis of their signal chains. Most important within the metabolic physiology are the biologically active secondary metabolites of myxobacteria and their molecular mechanisms of action. The global distribution of myxobacteria in soils is described on the basis of data given in the literature as well as of comprehensive analyses of 1398 soil samples from 64 countries of all continents. The results are analyzed with respect to the spectrum and number of species depending on ecological and habitat-specific factors. The myxobacterial floras of different climate zones are compared. Included are myxobacterial species adapted to extreme biotopes. The efficiency of different methods used presently for isolation of myxobacteria is compared.

The taxonomic composition of denitrifying bacteria in soddy podzolic soil was studied by the succession analysis method. This method revealed a significant variation in the taxonomic composition of denitrifying microorganisms in the course of succession. In contrast to succession analysis, the single microbiological analysis of soil samples reflected only the late stage of succession and thus led to an underestimation of the major members of succession. Myxobacteria were found to be the most active denitrifiers at the early stages of succession, whereas bacilli dominated at its late stages. The bacilli were represented by three facultatively anaerobic species, Bacillus cereus, Bac. circulans, and Bac. polymyxa.

In order to determine whether morphological criteria are suitable to affiliate myxobacterial strains to species, a phylogenetic analysis of 16S rDNAs was performed on 54 myxobacterial strains that represented morphologically 21 species of the genera Angiococcus, Archangium, Chondromyces, Cystobacter, Melittangium, Myxococcus, Polyangium and Stigmatella, five invalid species and three unclassified isolates. The analysis included 12 previously published sequences. The branching pattern confirmed the deep trifurcation of the order Myxococcales. One lineage is defined by the genera Cystobacter, Angiococcus, Archangium, Melittangium, Myxococcus and Stigmatella. The study confirms the genus status of 'Corallococcus', previously 'Chondrococcus', within the family Myxococcaceae. The second lineage contains the genus Chondromyces and the species Polyangium ('Sorangium') cellulosum, while the third lineage is comprised of Nannocystis and a strain identified as Polyangium vitellinum. With the exception of a small number of strains that did not cluster phylogenetically with members of the genus to which they were assigned by morphological criteria ('Polyangium thaxteri' Pl t3, Polyangium cellulosum ATCC 25531T, Melittangium lichenicola ATCC 25947T and Angiococcus disciformis An d1), the phenotypic classification should provide a sound basis for the description of neotype species in those cases where original strain material is not available or is listed as reference material.

In an attempt to isolate indigenous marine myxobacteria from coastal samples, we obtained two swarm forming bacteria. Both isolates formed cell aggregates which, at least in one isolate, developed to fruiting body-like structures consisting of a mass of myxospore-like cells. The optimum NaCl concentrations for their growth were between 2 and 3%, comparable to the NaCl concentration of seawater. This growth characteristic strongly suggests that the two isolates are specific marine bacteria. The 16S rDNA sequence studies indicated that the two isolates were related to the genus Nannocystis. Based on the phylogenetic distances between branches, we concluded that the isolates should be assigned to two new myxobacterial genera.

The ability of bacterial organisms to produce an extracellular polysaccharide matrix known as slime has been associated with increased virulence and delayed infections in various prosthetic implants. Within a biofilm, this slime may protect the embedded bacteria from host defense mechanisms, especially phagocytosis by polymorphonuclear leukocytes. To determine whether planktonic Staphylococcus epidermidis is protected in a similar way, a novel flow cytometric assay was performed, measuring ingestion and adherence during phagocytosis and the production of superoxide during oxidative burst. Hydrophobicity was determined by hydrophobic interaction chromatography. Slime-producing S. epidermidis RP62A and its phenotypic variant, non-slime-producing RP62A-NA, were compared. The results showed increased phagocytosis of RP62A at 2, 5, 10, and 30 min; increased adherence of RP62A at 30 s and 30 min; and increased superoxide production of RP62A after 2 min. Decreased hydrophobicity of RP62A over RP62A-NA was correlated with a hydrophilic slime coat. The data argue that the host aggressively combats slime-producing S. epidermidis. This biological phenomenon is potentially important during bacteremia to prevent further adhesion, accumulation, and the genesis of a bacterial biofilm on implants or tissue surfaces.

Seven strains of the myxobacterium Chondromyces crocatus, isolated from widely separated geographic regions, were investigated for the presence of an associate gram-negative, rod-shaped companion bacterium that is phylogenetically related to the genus Sphingobacterium and has been named "Candidatus comitans" (C. A. Jacobi, E. Stackebrandt, H. Reichenbach, and B. J. Tindall, Int. J. Syst. Bacteriol. 46:119-122, 1996). Five of the Chondromyces strains were found to be associated with a companion bacterium, and one strain lost its companion during the study. A 16S ribosomal DNA (16S rDNA) clone library was generated for each Chondromyces culture. Sequence similarity was > 99.1% for all but one strain of C. crocatus and all but one strain of "Candidatus comitans". The three analyzed 16S rDNA clone sequences of the companion of Cm c7 indicated that this companion strain is slightly less related to the other companion strains. The association between the companion and the myxobacterium including the sporangioles was determined by in situ hybridization with fluorescently labeled rRNA probes and scanning confocal laser microscopy. Based on these results, there are indications that the companion strains may survive environmental stress by inclusion in the aggregates and in the sporangioles of the myxobacterium.

Twenty-eight myxobacterial strains, representing members from all three subgroups, were screened for the presence of retron elements, which are novel prokaryotic retroelements encoding reverse transcriptase. The presence of retrons was determined by assaying strains for a small satellite DNA produced by reverse transcription called multicopy, single-stranded DNA (msDNA). An msDNA-producing retron appeared to be absent from only one of the strains surveyed. DNA hybridization experiments revealed that retron elements similar to retron Mx162, first identified in Myxococcus xanthus, were found only among members of the Myxococcus subgroup; that is, each of the seven different genera which constitute this subgroup contained a Mx162 homolog. Another retron element also appeared to have a clustered distribution, being found exclusively within the Nannocystis subgroup of the myxobacteria. A retron element of the Mx162 type was cloned from Melittangium lichenicola, and its DNA sequence was compared with those of similar elements in M. xanthus and Stigmatella aurantiaca. Together, the degree of sequence diversity, the codon bias of the reverse transcriptase genes, and the clustered distribution of these retrons suggest a possible evolutionary scenario in which a common ancestor of the Myxococcus subgroup may have acquired this retroelement.

The primary sequence and secondary structural features of the 16S rRNA were compared for 12 different myxobacteria representing all the known cultivated genera. Analysis of these data show the myxobacteria to form a monophyletic grouping consisting of three distinct families, which lies within the delta subdivision of the purple bacterial phylum. The composition of the families is consistent with differences in cell and spore morphology, cell behavior, and pigment and secondary metabolite production but is not correlated with the morphological complexity of the fruiting bodies. The Nannocystis exedens lineage has evolved at an unusually rapid pace and its rRNA shows numerous primary and secondary structural idiosyncrasies.

Novel citrate-hydroxamate siderophores, named nannochelins A, B and C, were isolated from the culture broth of the myxobacterium Nannocystis exedens strain Na e485. The new substances showed weak growth-inhibitory activity against some bacteria and fungi.

Myxobacteria are soil bacteria whose unusually social behavior distinguishes them from other groups of procaryotes. Perhaps the most remarkable aspect of their social behavior occurs during development, when tens of thousands of cells aggregate and form a colorful fruiting body. Inside the fruiting body the vegetative cells convert into dormant, resistant myxospores. However, myxobacterial social behavior is not restricted to the developmental cycle, and three other social behaviors have been described. Vegetative cells have a multigene social motility system in which cell-cell contact is essential for gliding in multicellular swarms. Cell growth on protein is cooperative in that the growth rate increases with the cell density. Rippling is a periodic behavior in which the cells align themselves in ridges and move in waves. These social behaviors indicate that myxobacterial colonies are not merely collections of individual cells but are societies in which cell behavior is synchronized by cell-cell interactions. The molecular basis of these social behaviors is becoming clear through the use of a combination of behavioral, biochemical, and genetic experimental approaches.

A previous study has shown that non-anti-Pseudomonas aeruginosa antibiotics can modify the mucoid character, a major virulence factor in cystic fibrosis P. aeruginosa. The purpose of the present study was to investigate further this phenomenon using a quantitative approach on fresh sputum. A total of 0.1 to 0.5 ml of sputum were homogenized, decimally diluted, plated on three series of Difco Pseudomonas isolation agar containing no antibiotics, roxithromycin (16.0 microgram/ml) or Rifampicin (16.0 microgram/ml) and incubated at 35 degrees C. In each of the three series, the cfus of PA by ml of sputum were evaluated after 24h, and the entire culture of plates with confluent growth was harvested after 96h and weighed on an analytical scale according to the correlation observed between the mucosity and the density of the culture. To date, 36 sputa from twenty-nine cystic fibrosis patients known to be chronically colonized by mucoid P. aeruginosa have been submitted to the protocol; the mean weight of control, roxithromycin and rifampicin cultures was respectively 3.12 +/- 1.62, 1.90 +/- 1.67 (p less than or equal to 0.0005) and 1.83 +/- 1.24 g (p less than or equal to 0.005) in parallel with mean cfu/ml of 2.24 +/- 6.45 x 10(8) (p less than 0.03) and 1.65 +/- 4.42 x 10(8) (p greater than 0.05); a more constant reduction (greater than or equal to 20%) of mucosity (expressed as the ratio of weight of antibiotic culture/control culture x 100) was observed with rifampicin (29 vs. 23/36 sputa), whereas a more drastic reduction (greater than or equal to 80%) was observed with roxithromycin (12 vs. 4).(ABSTRACT TRUNCATED AT 250 WORDS)

The gliding myxobacteria, common and easily obtainable soil organisms, exhibit a fascinating social organization which culminates in the formation of fruiting bodies by an ordered cooperation of hundreds of thousands of cells. Inside the fruiting body, the vegetative cells transform themselves into dormant myxospores. The myxobacteria have become potent model systems for studying morphogenetical problems at the prokaryotic organizational level, particularly since the methods of modern genetics now may be readily applied to them.

The first section of this review covers the important characteristics of the genera Cytophaga and Sporocytophaga. The topics discussed include vegetative cell structure, the spreading habit, and degradation of macromolecules. A historical account of these two genera follows, together with a discussion on the definition of, and species differentiation with the genus Cytophaga, and on the taxonomy of Sporocytophaga. The third section deals with the relationships of the cytophagas with the flavobacteria and includes a brief history of Flavobacterium, reassignation of some species to Cytophaga, differentiation from Cytophaga, and a discussion on the definition of the genus Flavobacterium. This is followed by a section dealing with the relationship of Cytophaga with the flexibacteria, starting with an introduction to the diversity of flexing organisms and taxonomic developments, and proceeding with the differentiation within the family Cytophagaceae, and species differentiation in Flexibacter. The concluding section includes a proposed redefinition of Cytophaga, a proposal regarding species conservation in this genus, and discussions on the relationship between the cytophagas and the myxobacteria and on the significance of cytophagas in the environment. The characteristics of all described species of Cytophaga, Flexibacter, and relevant flavobacteria are tabulated and a bibliography is presented.

The molar percentages of guanine plus cytosine in the DNA of 51 strains of Simonsiellaceae were determined by buoyant density ultracentrifugation of cell lysates in CsCl. The DNA base ratios ranged from 41-55 mole-% guanine plus cytosine. These values fall within the range known for the Order Cytophagales, the non-fruiting gliding bacteria, and are outside the range of the Order Myxobacterales, the fruiting myxobacteria. Among the strains of the genus Simonsiella, four distinct group can be delineated on the basis of source of origin (sheep, dog, cat, human) and GC content. The neotype of Alysiella filiformis has a GC content of 45.4 mole-%.

The indicator function of aquatic myxobacteria for the purpose of evaluating drinking water quality as well as their occurrence in sewage effluent make it desirable to describe these hitherto little known organisms in more detail. To this end, a comparative investigation of anaerobic myxobacteria of the genus Sphaerocytophaga, two typical representatives of the Order Myxobacterales (Myxococcus fulvus, Sporocytophage cauliformis), and a strain ov Vitrepscilla (Vitroescilla proteolytica) was undertaken. With respect to culture morphology, the migratory fringe surrounding colonies of Sphaerocytophaga similar to the other strains studies was a prominent characteristic. In particular, the similarity with colonies of Sporocytophaga cauliformis was apparent. The gliding motility typical of Sphaerocytophaga could be demonstrated in all of the strains investigated. Scanning electron micrographs revealed an amorphous layer of slime covering the cell surfaces in all strains compared in this study, thus excluding the existence of more rigid organelles of locomotion. Taxonomically, the anaerobec myxobacteria of the oral cavity (Sphaerocytophaga) belong to the Order "Myxobacterales" and not the "Eubacterales", i.e., to the genus Fusobacterium. This is clearly suggested by their motility lacking flagella and, above all, by their cell morphology which differs from the Eubacterales.