| Myxobacteria exhibit remarkably complex social behaviors, which are essential for their survival in natural environments (Shimkets 1990). Myxobacterial cells crawl on solid surfaces in swarms and feed cooperatively on macromolecules and on other microbial cells. When food is exhausted, hundreds of thousands of myxobacterial cells accumulate on solid surfaces to form multicellular fruiting bodies, inside which myxospores, which are resistant to adverse conditions, develop. Additionally, myxobacteria have the largest known genomes and the most complicated gene-regulation systems, which are probably necessary for their complex social lifestyle (Goldman et al.,2006; Zusman et al.,2007). Using specific identification techniques based on their social characteristics, such as the ability to form fruiting bodies and characteristic swarms, myxobacterial strains have been identified in various terrestrial habits (Reichenbach 1999, Dawid 2000). These cultured soil myxobacterial strains are normally unable to grow in a high-salt environment (Reichenbach 1999, Li et al.2002). However, some halophilic and halotolerant myxobacterial strains have been isolated from marine environments (Fudou et al. 2002, Iizuka et al.1998, Iizuka et al.2003a, Iizuka et al.2003b, Li et al.2002). Based on phylogenetic analyses and living patterns, the halophilic myxobacteria are considered indigenous to the ocean, whereas the halotolerant myxobacteria are probably soil myxobacterial strains that have adapted to oceanic conditions (Zhang et al.2005). Myxococcus fulvus HW-1 is a typical salt-tolerant marine strain that was isolated from a coastal sample (Li et al.2002). When cultured in a low concentration of saltwater, the strain exhibits a culture phenotype similar to soil myxobacterial strains, such as developing fruiting bodies and myxospores in response to starvation on solid surfaces. However, when the saltwater concentration is increased, the strain changes its living pattern by forming myxospores directly from vegetative cells (Zhang et al.2005). These characteristics make HW-1 an ideal model for studying the adaptation mechanisms of salt-tolerant myxobacteria to marine environments. Bacteria are single-cell organisms and normally live as individuals. In some complicated cases, social behaviors are also present, such as the formation of biofilms, quorum-sensing behaviors, and development of multi-cellular fruiting body structures resistant to adverse natural environments. In such cases, cells need to recognize, communicate and interact with each other to realize the social lifecycle in the natural environments. Myxobacteria are unique among the prokaryotes in their complicated multi-cellular social lifecycle. The sociality of myxobacteria is species-specific. For example, the fruiting bodies are genus-or species-specific, as no chimera composed of cells of different myxobacterial species has been found in natural or laboratory conditions (Smith and Dworkin,1994). The swarming colonies of different myxobacterial species or even different strains of one species can grow separately when closely pair-inoculated on an agar plate (Fiegna and Velicer,2005). However, competition, antagonism and even cannibalism can occur if two close relatives of myxobacteria, such as different species of Myxococcus or different lineages of M. xanthus are mixed (Smith and Dworkin,1994; Fiegna and Velicer,2005). In contrast, if two distantly related species are mixed, the myxobacterial cells find others of their own species, grow separately and develop their own colonies. Thus, myxobacteria exhibit the evolutionary adaptations of cell-cell recognition, competition and cooperation in addition to the adaptation of soil myxobacterial strains to oceanic conditions.There is much interest in understanding how myxobacteria and their social behaviors have adapted to natural environments. Therefore, this study was carried out as follows:(I) The adaptation mechanisms of soil myxobacterial strains to oceanic conditions were studied using the halotolerant strain HW-1 as a model strain. First, to study the relationship between behavioral shifts and the adaptation to oceanic conditions, the HW-1 strain was randomly mutagenized using transposon insertion. From more than 300 transformants, two strains that were able to grow in a completely dispersed state in liquid CTT broth were obtained. One of the two mutants, designated YLH0401, was chosen for further study. The mutant did not develop fruiting bodies or myxospores, was deficient in S-motility, produced less extracellular matrix, and was less salt-tolerant. YLH0401 was determined to be mutated by a single insertion in a large gene of unknown function (7,011 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. The gene is conserved in some salt-tolerant myxococcus strains but does not exist in the type strains of myxococcus that were obtained from soil samples. The profound phenotypic changes produced by mutation of this gene suggest it has important roles in maintaining salt tolerance and social behavior in marine environments. This horizontally transferred gene may allow Myxococcus to adapt to oceanic conditions. The marine salt-tolerant myxococcus strains probably horizontally exchange genes to maintain and reinforce social behavior to adapt to oceanic conditions.Second, based on chip-hybridization results revealing that the expression of 35 two-component genes in the salt-tolerant strain HW-1 were significantly modified (mostly down-regulated) by the presence of seawater, the roles of seawater-regulated two-component-related genes in the behavioral shifts of HW-1 were analyzed. Sequencing the seawater-regulated genes in HW-1 revealed that they are highly similar to their homologues in DK1622, suggesting they have similar functions in both strains. Next, the homologues of 23 seawater-regulated two-component-related genes in DK1622 were knocked out to analyze their functions in response to changes in salinity. Om031 (MXAN3106) had the most significant change in expression (down-regulated) in response to seawater, whereas Tc105 (MXAN4042) had the greatest increase in expression. Therefore, the functions of these two genes were analyzed first. In addition to having increased salt tolerance, sporulation of the MXAN3106 mutant was enhanced compared to DK1622, whereas mutating MXAN4042 produced the opposite result. These results indicate that the two seawater-regulated genes played important roles in the adaptation of HW-1 to oceanic conditions. The functions of the other 21 genes in the behavioral shifts of HW-1 were analyzed further. The results indicated that 10 of the 21 genes played roles in the seawater adaptation. Based on the results of chip-hybridization and gene function analysis, we concluded that increasing and decreasing the expression levels of these multifunctional genes allows cells to quickly respond and efficiently acclimate to changing conditions in different environments.The chip-hybridization method facilitated the high-throughput analysis of genes predicted to play important roles in seawater adaptation. This method overcomes the research obstacle of the low transformation efficiency of HW-1. However, random mutagenizing transposon insertion made it possible to detect genes specific to the salt-tolerant strains. In addition to the horizontal exchange of genes to maintain and reinforce social behaviors necessary for adapting to oceanic conditions, the marine salt-tolerant Myxococcus strains probably also up-or down-regulate the expression of multifunctional genes at the transcriptional level to quickly and efficiently respond and acclimate to environmental changes.(II) Social motility is important for the social behavior of Myxococcus. Social motility depends on type IV pili, which are composed of pilin monomers. The pilin monomer is the translational product of the pilA gene. Velicer reported that most of his artificial evolution mutants mapped to the pili locus (Velicer,2002). Sequence analysis indicated that the conserved 5'nucleotides and changeable 3'nucleotides constitute the pilA gene. The changeable 3'bases suggested that the pilA gene had diversely evolved in natural environments. Perhaps there is a relationship between pilA diversification and cell-cell recognition and communication. The pilA gene was selected for further research. The sequence diversity of the pilA gene in myxococcus strains was analyzed first. pilA genes from 59 myxococcus strains were amplified using different PCR methods. Phylogenetic analysis indicated that the sequences of the 59 myxococcus strains could be divided into 15 clades, which were separated by the differences in their amino acid sequences. Then, eight sequence-varied pilA genes from the 15 clades were cloned into the automatically replicating plasmid pZJY41 and introduced into the pilA-deficient mutant DK10410 of M. xanthus DK1622. The social behaviors of the chimeras were studied. With the foreign pilA genes, most of the chimeras recovered the S motility, even though the introduced PilA proteins had substantially different C-termini. The foreign pilA gene in DK10410 caused the cells to resume EPS production, providing experimental evidence supporting the hypothesis of Yang (Black,2006). Our results suggest that the social motility mechanism of myxococcus in natural environments is similar to the model strains.The function of pili in cell-cell recognition during nutritional swarming and fruiting body formation was analyzed further using the foreign pilA-restored strains. The role of pili in cell-cell recognition during nutritional swarming was analyzed first. Interestingly, when the foreign pilA-restored Myxococcus cells were closely pair-inoculated with their parent strains, i.e., M. xanthus DK1622 and the pilA-donor lineages, they could not merge with each other, but rather formed separate colonies; conversely, the colonies of the pilADK1622-restored cells merged with colonies of DK1622 but not the pilA-restored chimeras. Next, the role of pili in cell-cell recognition during fruiting body formation was analyzed. Chimeric fruiting bodies were formed by the cells of different myxobacterial species when the foreign pilA-expressing Myxococcus cells were mixed with DK1622. However, the results were varied when the foreign pilA-expressing Myxococcus cells were mixed with their parent strains. Some formed chimeric fruiting bodies, some formed two distinct fruiting bodies, and with others, only one of the two mixed strains formed fruiting bodies. Our results indicate that the pili play a role in cell-cell recognition. S motility of Myxococcus cells is motored by the pilus, a polymer of the PilA protein. The pilus protrudes from the front of the cell, recognizes and binds to certain substrates, and then contracts to pull cells forward. Therefore, the diverse evolution of the pilA gene is important for the evolution of cell-cell recognition and communication of myxococcus in natural environments.
|
PDF Full Text Request