The Cell Behavior And Its Molecular Mechanism Of Marine Myxobacteria

The myxobacteria are considered to be the typical soil microorganisms and very common in many terrestrial environments. Myxobacterial cells move by gliding in swarms on solid surfaces, feed by cooperatively digesting macromolecules as well as whole microorganisms, and form multicellular fruiting bodies on solid surfaces. The resting cells, or myxospores, develop within fruiting bodies. Fruiting bodies only develop on solid surfaces and terrestrial myxobacteria do not usually tolerate greater than 1% NaCl. Although myxobacteria could grow in liquid broth, but it never be verified that rest forms of myxobacteria could develop in water. Since myxobacteria were occasionally isolated from aqueous environments, Reichenbach and Dawid considered the myxobacteria isolates from water were the rest forms-myxospores in fruiting bodies which were flushed into the water and germinated.On the other hand, myxobacteria could be isolated from marine samples, as proven by the 16S rDNA fragments amplified from marine samples and permanently cold marine sediments. In 1998 Iizuka et al isolated two halophilic marine myxobacteria. The 16S rDNA sequence studies indicated that the two isolates were related to the genus Nannocystis. The closest relative for strain SHI-1 was N. exedens (level of similarity 89.3%), and those for strain SMP-2 were N. exedens (level of similarity 83.2%). Based on the phylogenetic distances between branches, the isolates were assigned to two new myxobacterial genera. With a special method, we also isolated some halotolerant myxobacteria strains from marine sample. Since Halophilic and halotolerant myxobacteria have been isolated from various marine samples, e.g. seawater. marine sands, mud, or sediments on marine animals and plants, which suggests that myxobacteria might be indigenous to the ocean. However, the natural marine environments do not favor the morphogenesis of the fruiting body structure, which not only protects myxospores but ensures the cell density for a new life cycle. So, how could these marine myxobacterial isolates live in the ocean?In this paper, we reported the different characteristics of halotolerant myxobacteria in terrestrial versus marine conditions. A halotolerant myxobacterial strain Myxococcus fulvus HW-1 (ATCC BAA-855) and a halophilic myxobacterial strain Haliangium ochraceum SMP-2 were employed as moaeis to study the growth and development characteristics in seawater or in fresh water, cellular density-dependentgrowth and the spherical cells characteristics of the marine myxobacteria. From the results obtained, we suggest the possible living patterns of myxobacteria in the ocean for the first time which could provide a clue for the illumination of the myxobacteria's evolution.Halotolerant Myxococcus strains HW-k 128^ 125-1 and 125-10-1 were ail isolated from marine samples, among them HW-1 and 125-10-1 could grow bearing the salinity in seawater(3.6%) . To perform the classic morphological classification, we observed the morphological characteristics of these four strains, including the shape and size of vegetative cell, myxospores and fruiting bodies and the configuration of swarms and pigments etc. To determine the G+C mol%, we analyzed the conditions and products by chemical degradation (perchloric acid and formic acid) and enzymatic degradation (phasphodiesterase I and bovine intestinal mucosa alkaline phophatase) of bacterial genome DNA with RP-HPLC, and established a systematic method to precisely measure the G+C mol% of bacterial DNA. 16S rDNA fragments of four strains were amplified, and PCR products were sequenced to make the phylogenetic analysis. The Genbank accession numbers of these four halotolerant strains were HW-1 (AF336801), 125-10-1 (AY072740), 128-7(AY032879) and 125-1 (AF466191). According to Bergey's Manual of Systematic Bacteriology (9th Edition) and The Prokaryote (2nd edition), the four halotolerant myxobacteria were classified as Myxococcales - Cystobacterineae - Myxococcaceae - Myxococcus fulvus HW-1, 128-7 and 125-1 and Myxococcus macrosporus 125-10-1.To study the four halotolerant Myxococcus strains about the growth and development characteristics in seawater or fresh water, and in different NaCl concentrations, either on agar or in liquid broth, the Haliangium ochraceum SMP-2 was employed as contrast. M. fulvus HW-1 was chosed as the halorotant strain model to the study, including culture characteristics, cellular density-dependent growth and the spherical cells characteristics involved SMP-2 checked in parallel.Myxococcus fulvus strain HW-1 grew in the medium ranging from 0% (DVY) to 130% seawater (The salinity was assayed to be 4.7%). The optimal growth conditions were at 0-80% seawater (DVY and 10-80% HVY. The salinity in 80% HVY is 2.9%). In DVY or low-seawater HVY, the swarms on agar or cell clumps in liquid were bright yellow. If the concentration of seawater was more than 70%, the color of swarms or clumps changed to light tan. To make a comparison, HW-1 and SMP-2were both inoculated onto VY/2 agar supplemented with different concentrations of sodium chloride (NaCl). Just as Iizuka described, Haliangium ochraceum strain SMP-2 is an obligate halophile. The strain was unable to grow without NaCl. The NaCl concentration required for growth ranged from 0.2% to 5%, with an optimal concentration between 1% and 3%. On the other hand, Myxococcus fulvus strain HW-1 could only tolerate NaCl concentration as high as 3% with an optimal concentration at 0-1.5%. HW-1 cells were greatly clumped when growing in liquid DVY or low seawater HVY, but much homogenized at high seawater concentration. In contrast. SMP-2 grew in tight clumps at all tested salt concentrations.The length of HW-1 cells changed in different growth stages, both in liquid and on agar. Besides, in response to seawater concentration, the cells also changed their size. However, after growth, almost all the cells, whether initially long or short, in liquid or on agar. shortened into spheres or ovoids, similar in size (1.2-1.8 urn in diameter) to the myxospores from the fruiting bodies. By contrast, SMP-2 had essentially no change of its cell size in response to different seawater concentrations. In the fruiting body-like structure of SMP-2 on agar, or in aged liquid culture, the cells were shortened into ovoids.The fruiting body formation of halotolerant HW-1 only occurred on solid surfaces, such as agar. and was strongly affected by the salinity. On DVY-agar or less than 60% HVY-agar. typical fruiting bodies, which were orange red and 100-150 urn in diameter with a cushion-like pedicel, appeared on the third day after inoculation. As the salinity increased, the fruiting body formation was delayed and the final structure became more rudimentary. On media containing 2.9% salinity (80% HVY-agar) or higher, the cells failed to produce fruiting bodies, but still aggregated to form multicellular mounds.To discuss the effects of the salts in seawater on growth rate and the development of fruiting bodies, morphogenesis of the fruiting bodies on VY/2 with different combination of the three main salts in seawater, i.e. NaCl, MgCh and CaCh was obsen'ed. Among the three salts, MgCb stimulated the growth and the fruiting body formation while the other two salts inhibited the process.In comparison, the halophilic myxobacterium SMP-2 was able to form a few fruiting body-like structures in the centre of the swarm. The structure changed a littlein response to the shift in salinity. However, if the seawater concentration in medium was 20% or lower, the cells seldom developed.The spherical cells of HW-1 from agar or liquid containing high seawater could survive heat-treatment at 60°C for 5min. The spherical cells from seawater media, whether in liquid or on agar, tolerated higher temperature than the myxospores developed in the fruiting bodies on DVY. The spherical cells from liquid DVY, or low seawater HVY died after 5 min of treatment at only 45 °C. The HW-1 myxospores from fruiting body and the spherical cells from liquid HVY with high seawater were able to germinate after at least five years' storage, while the spherical cells from liquid DVY died after 1 month. The results strongly support that HW-1 is able to develop myxospores without morphogenesis of fruiting bodies in seawater conditions, while the spherical cells formed in liquid DVY were only degenerated vegetative cells. The resting form of HW-1 in terrestrial conditions was the fruiting body structure developed on solid surfaces.SMP-2 developed a few fruiting body-like structures on the agar. The transformative cells in the fruiting body-like structure of SMP-2 were able to tolerate 5 min treatment at 55-60 °C. On the other hand, the cells of SMP-2 in liquid also transform their shape and size in old cultures, just like the case of the cells developing in the fruiting body-like structure, and these transformative cells in liquid culture were able to tolerate 55-60 °C heat treatments. That is to say, the transformative cells of SMP-2 in old liquid culture or in fruiting body-like structure on agar were real myxospores.HW-1 vegetative cells from DVY or 10% HVY formed 0-2 colonies if 0.2ml of lxlO4 cell/ml suspension was spread on a 90mm diameter plate, whether on DVY, 50% HVY or 100% HVY-agar, or were able to grow if lxlO4 cells per ml were inoculated into liquid media. Inoculating 0.2ml of lxlO3 cells/ml on a plate did not form colonies. However, the cells from higher seawater-containing liquid HVY (20% or higher) were able to grow and form swarms on a 90 mm diameter plate with only 0.2ml of 10 cells/ml suspension (less than 10 cells per plate) or at the concentration of 10 cells/ml of inoculum in liquid media, DVY, 50% HVY, or 100% HVY.On the other hand, the halophilic strain SMP-2 cultured*in HVY containing different seawater, whether in liquid or on agar, could form swarms on a 90mmdiameter plate with a concentration of about 1 x 105 cells per plate, but failed with lxlO4 cells per plate. That is to say, growth of strain SMP-2 is strongly dependent on cell density.We suggest that there are two growth strategies for myxobacteria. One is simple -forming myxospores directly from vegetative cells - and the cells grow independent of cell-density. The other is complicated - forming fruiting body structures from numerous cells - and the cellular growth is dependent on cell-density. A fruiting body ensures that a new life cycle is able to start in a terrestrial environment. It seems that the adaptation of these halotolerant myxobacteria to a marine environment is a degenerative procedure. The two living patterns could shift if the cells migrate between the two environments.Halicmgium ochaceum SMP-2, a halophilic myxobacterium uses a different living strategy. SMP-2 grew in tight clumps at all salt concentrations, and its growth was greatly dependent on cell-density. The cells rest on solid media by constructing fruiting body-like structures containing myxospores or in liquid by directly developing myxospores. Up to now, all the halophilic myxobacterial strains are phylogenetically assigned to novel genera, such as Haliangium ochraceum, Plesiocysiis pacifica, Enhygromyxa salina. Considering that there have been no closely related myxobacteria found in soil yet, it is suggested that the halophilic myxobacteria might form a different evolutionary group that is indigenous to the ocean.As the use of plasmid derived from Tn5, transposon mutation was the major method to get Myxococcus xanthus mutants. To illuminate the genetic mechanism of Myxococcus xanthus special microbiological characteristics especially the salt-tolerance, we used PI'. ITn5 lac to introduce transposon Tn5 lac into Myxococcus fulvus HW-1 chromosome and established a mutation library of about 200 mutants. We selected four salt-sensitive mutants from the bank and validated the Tn5 lac insertion in such three ways below: Southern blotting - a 16 kb hybridizition signal from the mutation chromosome; the mutants were Kmr and could express the lacZ gene to produce ($- ga/actosidase; using Tn5 lac sequence to design primers, and amplified PCR with the mutatants chromosome DNA templates, we could get the target gene sequences of Tn5 and lacZ, but no products from HW-1 chromosome.We have examed the phonotype of the four mutants and determined the effects of Tn5 lac insertion on mutants cell behaviors such as growth development and motility et al and qualified the salt-tolerance decrease of the mutants. Mutants strains 2TD96 and 2TD108 could only grow in the condition of below 70% seawater concentration and 2.0% NaCl and could not form fruiting bodies. 2TD87 and 2TD120 could only grow in the condition of below 80% seawater concentration and 2.0% NaCl and could form fruiting bodies only under several salinity conditions. As determined the lacZ gene product -P-ga/actosidase activity, we found that lacZ gene could only be expressed in development stage. These results clewed that the gene involved in the development could be correlative to the salt-tolerance.Using TAIL-PCR. we isolated the flanking region fragments of Tn5 lac insertion about 1.5 kb. Sequencing analysis showed that Tn5 specific sequence was on one end. Bioinformatics analysis showed that function of this gene fragment could be related to several kinds of kinase in sigal transduction pathway. This would be the foundation to illuminate the halotorant Myxococcus salt-tolerance molecular mechanism.