Genomic Analysis of Bacillus megaterium NCT-2 Reveals Its Genetic Basis for the Bioremediation of Secondary Salinization Soil

Bacillus megaterium NCT-2 is a nitrate-uptake bacterial, which shows high bioremediation capacity in secondary salinization soil, including nitrate-reducing capacity, phosphate solubilization, and salinity adaptation. To gain insights into the bioremediation capacity at the genetic level, the complete genome sequence was obtained by using a multiplatform strategy involving HiSeq and PacBio sequencing. The NCT-2 genome consists of a circular chromosome of 5.19 Mbp and ten indigenous plasmids, totaling 5.88 Mbp with an average GC content of 37.87%. The chromosome encodes 5,606 genes, 142 tRNAs, and 53 rRNAs. Genes involved in the features of the bioremediation in secondary salinization soil and plant growth promotion were identified in the genome, such as nitrogen metabolism, phosphate uptake, the synthesis of organic acids and phosphatase for phosphate-solubilizing ability, and Trp-dependent IAA synthetic system. Furthermore, strain NCT-2 has great ability of adaption to environments due to the genes involved in cation transporters, osmotic stress, and oxidative stress. This study sheds light on understanding the molecular basis of using B. megaterium NCT-2 in bioremediation of the secondary salinization soils.

Orthologues ofBacillus subtilisSpore Crust Proteins Have a Structural Role in theBacillus megateriumQM B1551 Spore Exosporium

ABSTRACTThe exosporium ofBacillus megateriumQM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of theBacillus megateriumexosporium. Here, we provide evidence that pBM600-encoded orthologues of theBacillus subtilisCotW and CotX proteins, which form the crust layer in spores of that species, are structural components of theBacillus megateriumQM B1551 spore exosporium. The introduction of plasmid-bornecotWand orthologouscotXgenes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in theBacillus megateriumQM B1551 exosporium.IMPORTANCEWhen starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, inBacillus megateriumQM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenicBacillus cereusfamily. In this work, we provide evidence that structurally important protein constituents of theBacillus megateriumexosporium are different from those in theBacillus cereusfamily. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.

Discovery of the Autonomously Replicating Plasmid pMF1 from Myxococcus fulvus and Development of a Gene Cloning System in Myxococcus xanthus

ABSTRACT 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 (∼105 CFU/μg 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.

Comparative Analysis of Physical Maps of Four Bacillus subtilis (natto) Genomes

ABSTRACT The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure. Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed. No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B. subtilis strain Marburg 168. However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SPβ, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types. The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.

Isolation and characterization of mutants of the citrus canker pathogen Xanthomonas axonopodis pv. citri that induce a distinct pattern of disease

Random mutagenesis with the transposon Tn5tac1 in Xanthomonas axonopodis pathovar citri, the causal agent of citrus canker, generated four mutants with altered pathogenicity. These mutants were classified into three groups: (i) the nonpathogenic (NP) mutants XT10 and XT122, which did not induce any visible symptoms in the host; (ii) the WS- mutant XT27, which induced a callus-like lesion but not a watersoaked lesion; and (iii) the CL- mutant XT37, which was unable to induce a callus-like eruption but did induce the formation of a watersoaked lesion around the infection site. The NP mutants failed to grow in planta, whereas the WS- and CL- mutants showed a reduced growth rate relative to that of the wild type. Co-inoculation of leaves with the WS- and CL- mutants did not result in complementation of their respective defects. The extent of extracellular accumulation of polysaccharide, protease, and amylase activities by each of the mutants was similar to that of the wild type. The extracellular activity of polygalacturonate lyase of XT27 was reduced relative to that of the wild type and other mutants. Unlike the wild type and other mutants, XT27 also required glutamic acid for growth in culture. Southern blot hybridization revealed that each of the mutants resulted from transposon insertion at a single site; the insertion sites for XT10 and XT27 were located in the chromosome, whereas those for XT37 and XT122 were located in the indigenous plasmids. These results provide evidence that bacterial genes contribute independently to the pathogenesis of citrus canker.Key words: citrus canker, pathogenicity genes, transposon mutagenesis.