Molecular cloning and biochemical characterization of a trehalose synthase from Myxococcus sp. strain V11.

The tresI gene of Myxococcus sp. strain V11 was cloned, and found to encode a trehalose synthase comprising 551 amino acids. The deduced molecular weight of the encoded TreS I protein 64.7 kDa and the isoelectric point (pI) was predicted to be 5.6. The catalytic cleft consists of the Asp202-Glu244-Asp310 catalytic triad and additional conserved residues. The recombinant (His)6-tag enzyme was expressed in Escherichia coli BL21(DE3) and purified by Ni2+-affinity chromatography, resulting in a specific activity of up to 172.7 U/mg. TLC and HPLC results confirmed that rTreS I can convert maltose into trehalose, with a yield of 61%. The KM and Vmax values of recombinant TreS I for maltose were 0.62 mM and 25.5 mM min-1 mg-1 protein, respectively. TreS I was optimally active at 35° and stable at temperatures of <25 °C. TreS I was stable within a narrow range of pH values, from 6.0 to 7.0. The enzymatic activity was slightly stimulated by Mg2+ and strongly inhibited by Fe3+, Co2+ and Cu2+. TreS I was also strongly inhibited by SDS and weakly by EDTA and TritonX-100.

Insights into the persistence and phenotypic effects of the endogenous and cryptic plasmid pMF1 in its host strain Myxococcus fulvus 124B02.

Many endogenous plasmids carry no noticeable benefits for their bacterial hosts, and the persistence of these 'cryptic plasmids' and their functional impacts are mostly unclear. In this study, we investigated these uncertainties using the social bacterium Myxococcus fulvus 124B02 and its endogenous plasmid pMF1. pMF1 possesses diverse genes that originated from myxobacteria, suggesting a longstanding co-existence of the plasmid with various myxobacterial species. The curing of pMF1 from 124B02 had almost no phenotypic effects on the host. Laboratory evolution experiments showed that the 124B02 strain retained pMF1 when subcultured on dead Escherichia coli cells but lost pMF1 when subcultured on living E. coli cells or on casitone medium; these results indicated that the persistence of pMF1 in 124B02 was environment-dependent. Curing pMF1 caused the mutant to lose the ability to predate and develop fruiting bodies more quickly than the pMF1-containing strain after they were subcultured on dead E. coli cells, which indicated that the presence of pMF1 in M. fulvus 124B02 has some long-term effects on its host. The results provide some new insights into the persistence and impacts of cryptic plasmids in their natural bacterial cells.

A Post-segregational Killing Mechanism for Maintaining Plasmid PMF1 in Its Myxococcus fulvus Host.

Although plasmids provide additional functions for cellular adaptation to the environment, they also create a metabolic burden, which causes the host cells to be less competitive with their siblings. Low-copy-number plasmids have thus evolved several mechanisms for their long-term maintenance in host cells. pMF1, discovered in Myxococcus fulvus 124B02, is the only endogenous autonomously replicated plasmid yet found in myxobacteria. Here we report that a post-segregational killing system, encoded by a co-transcriptional gene pair of pMF1.19 and pMF1.20, is involved in maintaining the pMF1 plasmid in its host cells. We demonstrate that the protein encoded by pMF1.20 is a new kind of nuclease, which is able to cleave DNA in vitro. The nuclease activity can be neutralized by the protein encoded by pMF1.19 through protein-protein interaction, suggesting that the protein is an immune protein for nuclease cleavage. We propose that the post-segregational killing mechanism of the nuclease toxin and immune protein pair encoded by pMF1.20 and pMF1.19 is helpful for the stable maintenance of pMF1 in M. fulvus cells.

Genetic and Functional Analyses of the DKxanthene Biosynthetic Gene Cluster from Myxococcus stipitatus DSM 14675.

DKxanthenes are a class of yellow secondary metabolites produced by myxobacterial genera Myxococcus and Stigmatella. We identified a putative 49.5 kb DKxanthene biosynthetic gene cluster from Myxococcus stipitatus DSM 14675 by genomic sequence and mutational analysis. The cluster was comprisedof 15 genes (MYSTI_06004-MYSTI_06018) encoding polyketide synthases, non-ribosomal peptide synthases, and proteins with unknown functions. Disruption of the genes by plasmid insertion resulted in defects in the production of yellow pigments. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis indicated that the yellow pigments produced by M. stipitatus DSM 14675 might be noble DKxanthene derivatives. M. stipitatus did not require DKxanthenes for the formation of heat-resistant viable spores, unlike Myxococcus xanthus. Furthermore, DKxanthenes showed growth inhibitory activity against the fungi Aspergillus niger, Candida albicans, and Rhizopus stolonifer.

Identification of the Phenalamide Biosynthetic Gene Cluster in Myxococcus stipitatus DSM 14675.

Phenalamide is a bioactive secondary metabolite produced by Myxococcus stipitatus. We identified a 56 kb phenalamide biosynthetic gene cluster from M. stipitatus DSM 14675 by genomic sequence analysis and mutational analysis. The cluster is comprised of 12 genes (MYSTI_04318-MYSTI_04329) encoding three pyruvate dehydrogenase subunits, eight polyketide synthase modules, a non-ribosomal peptide synthase module, a hypothetical protein, and a putative flavin adenine dinucleotide-binding protein. Disruption of the MYSTI_04324 or MYSTI_04325 genes by plasmid insertion resulted in a defect in phenalamide production. The organization of the phenalamide biosynthetic modules encoded by the fifth to tenth genes (MYSTI_04320-MYSTI_04325) was very similar to that of the myxalamid biosynthetic gene cluster from Stigmatella aurantiaca Sg a15, as expected from similar backbone structures of the two substances. However, the loading module and the first extension module of the phenalamide synthase encoded by the first to fourth genes (MYSTI_04326-MYSTI_04329) were found only in the phenalamide biosynthetic gene cluster from M. stipitatus DSM 14675.

Complete Genome Sequence and Comparative Genomics of a Novel Myxobacterium Myxococcus hansupus.

Myxobacteria, a group of Gram-negative aerobes, belong to the class δ-proteobacteria and order Myxococcales. Unlike anaerobic δ-proteobacteria, they exhibit several unusual physiogenomic properties like gliding motility, desiccation-resistant myxospores and large genomes with high coding density. Here we report a 9.5 Mbp complete genome of Myxococcus hansupus that encodes 7,753 proteins. Phylogenomic and genome-genome distance based analysis suggest that Myxococcus hansupus is a novel member of the genus Myxococcus. Comparative genome analysis with other members of the genus Myxococcus was performed to explore their genome diversity. The variation in number of unique proteins observed across different species is suggestive of diversity at the genus level while the overrepresentation of several Pfam families indicates the extent and mode of genome expansion as compared to non-Myxococcales δ-proteobacteria.

Advanced mutasynthesis studies on the natural α-pyrone antibiotic myxopyronin from Myxococcus fulvus.

Myxopyronin is a natural α-pyrone antibiotic from the soil bacterium Myxococcus fulvus Mx f50. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by binding to a part of the enzyme not targeted by the clinically used rifamycins. This mode of action makes myxopyronins promising molecules for the development of novel broad-spectrum antibacterials. We describe the derivatization of myxopyronins by an advanced mutasynthesis approach as a first step towards this goal. Site-directed mutagenesis of the biosynthetic machinery was used to block myxopyronin biosynthesis at different stages. The resulting mutants were fed with diverse precursors that mimic the biosynthetic intermediates to restore production. Mutasynthon incorporation and production of novel myxopyronin derivatives were analyzed by HPLC-MS/MS. This work sets the stage for accessing numerous myxopyronin derivatives, thus significantly expanding the chemical space of f α-pyrone antibiotics.

Exploration on the biotechnological aspect of the ureolytic bacteria for the production of the cementitious materials--a review.

Biomineralization is a process that leads to the formation of minerals using the biologically or biotechnologically mediated route. Calcium carbonate is one such biomineral that is secreted by the ureolytic bacteria which contributes for the strengthening and improvement of cementitious and sandy materials. It is a new and innovative area in the geotechnological engineering and structural engineering due to its wide range of implications in strengthening of soil, sand, stone, and cementitious materials. The shape and size of the calcium carbonate particle vary with the strain of the bacterium used, and it is species specific. This paper aims in the critical review of the mechanism of calcium carbonate precipitation by the bacterium, various bacteria involved, and the useful outputs of the technique of biomineralization. Based on the critical review, it also recommends the future development and research in the field to develop a technology that can strengthen the existing and the proposed structures.

Nonadaptive processes can create the appearance of facultative cheating in microbes.

Adaptations to social life may take the form of facultative cheating, in which organisms cooperate with genetically similar individuals but exploit others. Consistent with this possibility, many strains of social microbes like Myxococcus bacteria and Dictyostelium amoebae have equal fitness in single-genotype social groups but outcompete other strains in mixed-genotype groups. Here we show that these observations are also consistent with an alternative, nonadaptive scenario: kin selection-mutation balance under local competition. Using simple mathematical models, we show that deleterious mutations that reduce competitiveness within social groups (growth rate, e.g.) without affecting group productivity can create fitness effects that are only expressed in the presence of other strains. In Myxococcus, mutations that delay sporulation may strongly reduce developmental competitiveness. Deleterious mutations are expected to accumulate when high levels of kin selection relatedness relax selection within groups. Interestingly, local resource competition can create nonzero "cost" and "benefit" terms in Hamilton's rule even in the absence of any cooperative trait. Our results show how deleterious mutations can play a significant role even in organisms with large populations and highlight the need to test evolutionary causes of social competition among microbes.

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease.

The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.

Characterization of the partitioning system of Myxococcus plasmid pMF1.

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics.

Genome sequence of the halotolerant marine bacterium Myxococcus fulvus HW-1.

Myxococcus fulvus HW-1 (ATCC BAA-855) is a halotolerant marine myxobacterium. This strain exhibits complex social behaviors in the presence of low concentrations of seawater but adopts an asocial living pattern under oceanic conditions. The whole genome of M. fulvus HW-1 will enable us to further investigate the details of its evolution.

Hdsp, a horizontally transferred gene required for social behavior and halotolerance in salt-tolerant Myxococcus fulvus HW-1.

Myxococcus fulvus HW-1, a salt-tolerant bacterial strain, which was isolated from a coastal environment, changes its behavior with different salinities. To study the relationship between behavioral shifts and the adaption to oceanic conditions, the HW-1 strain was randomly mutagenized using transposon insertion, producing a dispersed-growing mutant, designated YLH0401. The mutant did not develop fruiting bodies and myxospores, was deficient in S-motility, produced less extracellular matrix and was less salt tolerant. The YLH0401 strain was determined to be mutated by a single insertion in a large gene of unknown function (7011 bp in size), which is located in a horizontally transferred DNA fragment. The gene is expressed during the vegetative growth stage, as well as highly and stably expressed during the development stage. This horizontally transferred gene may allow Myxococcus to adapt to oceanic conditions.

[Construction and application of plasmid pZCY11 for analyzing gene functions and expressions in Myxococcus].

OBJECTIVE: To construct a plasmid for analyzing gene functions and expressions and to study the MXAN1334 gene in Myxococcus xanthus with the plasmid. METHODS: We constructed the plasmid vector pZCY11, amplified MXAN1334 gene fragment from M. xanthus DK1622 by PCR, and inserted the fragment into a site upstream of lacZ, resulting in the recombinant plasmid pZCY13. The plasmid pZCY13 was transformed by electroporation to DK1622, producing a mutant ZC16-18 (deltaMXAN1334). RESULTS: The plasmid pZCY11 carried the resistance gene aph as the selectable marker, the replication origin of OriR6K and promoterless reporter gene lacZ. We examined the swarm expansions of ZC16-18 on CTT hard and soft agar, and the result indicated that MXAN1334 gene was probably involved in gliding motility in M. xanthus. In addition, beta-galactosidase activity of ZC16-18 was detected by X-gal assay and the blue color developed was used to mark the colony growth. Time of colour showed that MXAN1334 gene was expressed in the early stage in M. xanthus. CONCLUSION: The plasmid vector pZCY11 made it more convenient for the study on functions and the expressions of target gene in M. xanthus.

Improving cellular properties for genetic manipulation by dispersed growing mutagenesis in Myxococcus fulvus HW-1.

One of the key limitations to genetic manipulation in myxobacteria is that the cells grow in clumps in liquid. A salt-tolerant strain HW-1 of Myxococcus fulvus was treated with UV irradiation and produced a completely dispersedly growing mutant UV684. There were no significant differences between the parent HW-1 and the mutant UV684 in terms of salt-tolerant growth. The mutant UV684 and the parent strain had the similar abilities of the fruiting body formation and S motility. Interestingly, the mutant exhibited high transformation/transposition efficiency with 10(5)-10(6) colony-forming units per microg DNA, which was about 10(3)-10(5) fold higher than HW-1. The results indicate that the mutation that led to dispersed growth in the UV684 mutant strain had a few impacts on social behavior, but greatly facilitated molecular genetic manipulation.

Seawater-regulated genes for two-component systems and outer membrane proteins in myxococcus.

When salt-tolerant Myxococcus cells are moved to a seawater environment, they change their growth, morphology, and developmental behavior. Outer membrane proteins and signal transduction pathways may play important roles in this shift. Chip hybridization targeting the genes predicted to encode 226 two-component signal transduction pathways and 74 outer membrane proteins of M. xanthus DK1622 revealed that the expression of 55 corresponding genes in the salt-tolerant strain M. fulvus HW-1 was significantly modified (most were downregulated) by the presence of seawater. Sequencing revealed that these seawater-regulated genes are highly homologous in both strains, suggesting that they have similar roles in the lifestyle of Myxococcus. Seven of the genes that had been reported in M. xanthus DK1622 are involved in different cellular processes, such as fruiting body development, sporulation, or motility. The outer membrane (Om) gene Om031 had the most significant change in expression (downregulated) in response to seawater, while the two-component system (Tc) gene Tc105 had the greatest increase in expression. Their homologues MXAN3106 and MXAN4042 were knocked out in DK1622 to analyze their functions in response to changes in salinity. In addition to having increased salt tolerance, sporulation of the MXAN3106 mutant was enhanced compared to that of DK1622, whereas mutating gene MXAN4042 produced contrary results. The results indicated that the genes that are involved in the cellular processes that are significantly changed in response to salinity may also be involved the salt tolerance of Myxococcus cells. Regulating the expression levels of these multifunctional genes may allow cells to quickly and efficiently respond to changing conditions in coastal environments.

Reclassification of Myxococcus flavescens Yamanaka et al. 1990VP as a later synonym of Myxococcus virescens Thaxter 1892AL.

The taxonomic relatedness between the species Myxococcus flavescens and Myxococcus virescens was investigated. Literature data had already indicated the synonymy between the two species but this observation had not been formalized. Additional evidence that the two taxa represent a single species was provided by comparison of metabolic properties, cellular fatty acid profiles and from a DNA-DNA reassociation value of >80 %. Data from this study led to the proposal that M. flavescens should be reclassified as a later synonym of M. virescens.

Myxococcus-from single-cell polarity to complex multicellular patterns.

Myxococcus xanthus creates complex and dynamic multicellular patterns as it swarms. The cells have two polar gliding engines: pulling type IV pili at their leading pole and pushing slime secretory nozzles at their lagging pole. Evidence is presented that slime secretion is vital for cell survival and that the peptidoglycan/cytoskeleton serves as a template to keep both engines oriented in the same direction. Swarming requires that each cell periodically reverse its gliding direction. For the leading pole to become the trailing pole, old engines are inactivated at both ends while new engines are being created at both ends. Reversal is initiated by a small G-protein reversal switch; a pulse of frzE approximately P from a reversal clock triggers MglA to bind GTP. Mgl.GTP then recognizes the engines that are currently in use and inactivates both of them. Meanwhile, new engines appear as instructed by the template, and the cell starts to glide in the opposite direction.

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus.

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.