The Screening And Function Identification Of The Extracytoplasmic Function Sigma Factors In The Myxococcus Xanthus DZ2

The sigma （σ） in RNA polymerase （RNAP） ensure the recognition of specific promoter sequences and direct the RNA holoenzyme to bind with a target promoter, resulting in the expression of different genes in a cell at different stages and conditions. The muti-subunit DNA-dependnt RNAP is the most conserved primary regulation of gene expression in prokaryotae. Except for the primary σ factors that are essential for the surival of cell, many bacteria can also code alternative a factors that play important roles in a wide range of cell physiology and biochemistry, such as membrane stress, nutrition hunger response, development, differentiation, and so on. The most factors in alternative a factors are extracytoplasmic function sigma （ECF a） factors that are a class of regulatory proteins and have the most varied sequences. As one of the signal transduction pathways that sense and respond to changing environment in prokaryotes, ECF σ factors are generally regulated by cognate ECF anti-σ factors, Typical myxobacteria are a class of environmental bacteria that maily live in soil and widely distribute in nature. They are known for their complex life cycle, including the extend of the multi-nutrion-cell, the formation of fruiting body and the differentiation of spores. Myxobacteria have so far the largest genome in bacteria and code numerous ECF a factors. We speculate that ECF σ factors weight a lot in the adaption to the changing natural habitats and the life style for myxobacteria.We researched on the 31 putative ECF a factor and 14 predicted ECF anti-σ factors that have not been reported in detail.To investigate the role of the 45 ECF σ and ECF anti-σ factors in Myxococcus xanthus （M. xanthus）, we constructed 45 mutants with the deletion of one factors in M. xanthus DZ2. ECF a factors mainly respond to the environmental signals. Given that the habitats of myxobacteria are diversity, we chose to use temperature, pH, osmosis, oxidation and salt as environmental signals for screening at first. We compared the growth of DZ2 in different gradients of these conditions and chose the sub-optimal growth condition as the stress condition for screenging at last. The designed conditions are 26℃,35.5℃, pH6.4, pH8.8,3% mannitol,100 mM NaCl, and 5 mM H2O2Then, we compared the growth of DZ2 and 45 mutants with the knockout of one ECF σ/anti-σ factors in the desiged conditions. The growths of all the mutants had no significantly difference with DZ2 in the normal condtion, which was in accordance with the feature of ECF σ factors. However, many mutants grew slower than DZ2 in desiged condition, suggesting that the corresponding ECF a factors played roles in responding to these specific signals. In the rescreening, we found that the growth curves of 11 mutants were notably different with DZ2 under specific stress conditions. Six mutants among them, ΔMXAN₂395, ΔMXAN₃686, ΔMXAN₃959, ΔMXAN₄147, ΔMXAN4₃15, grew slower than DZ2 in only one specific stress condition. But the other 5 mutants, ΔMXAN₄148, ΔMXAN₆460, ΔMXAN₆461, ΔMXAN₇214 grew slower than DZ2 in multiple stress condtion, which indicated these one deleted gene was involved in responding to diverse stress.As we all know, myxobacteria are renowned for their ability to sporulate within fruiting bodies and form spores when the nutrition is limited. This progress is subjected to strictly controled in space and time, involed in complex regulatory network.and including many regulatory proteins, such as 2TCS. Except for starvation, are the developmental genes expression also be influenced by other unfavorable enviroment? Do the ECF a factors participate in the regulation of the fruiting body development? In order to answer these questions, we compared the motility. fruiting body development and the rate of sporulation of all the muntans and DZ2 under the condition of 0 mM H2O2 and 10 mM H2O2, respectively. To our surprise, there were 25 mutans that showed some difference with DZ2 in motility, only their adventure motility （A-motility） or social motility （S-motility） had been effcted for some of them, but both A and S are different for others. Apart from 5 mutants, most of the muttants showed slower motility under the condition of 10 mM H2O2 as compared with DZ2. However, the role of ECF σ factors in the development and sporulation cascade were more diversity. For example, there were 7 mutants showed some differnece with DZ2 after 5 days （120 h） in CFL culture, but another 5 mutants were able to dvelopment and form spores under the starvation condition. To our interst, when we imposed additional oxidative stress, the develoment of 3 mutants respectively with the deletion of MXAN₄316, MXAN₅410, MXAN₇214 delayed and their rate of sporulation decreased. Nevertheless, the other 2 mutants respectively with the knockout of MXAN₂394 and MXAN₄986 differed completely. Therefore, ECF a factors did play an extensive and complex role in the motility. fruiting body development and sporulation in M. xanthus.Since ΔMXAN₆461 grew slower than DZ2 in 35.5℃, pH 6.4, pH 8.8,3% mannitol,100 mM NaCl,5 mM H2O2, and couldn’t form mature spores. We were wery interested in the ECF factor MXAN₆461. Firstly, we constructed MXAN₆461_comp strain （the MXAN 6461 complementation strain）. We found that MXAN₆461_comp strain was able to restore growth to a level similar to that of DZ2 strain in these specific stresses. Hence, MXAN₆461 exactly were a broad spectrum ECF a factors, playing a crucial role in heat, acid, alkaline, salt and oxidative stress resistance in M. xanthus. The co-transcription of 6 genes, from MXAN₆457 to MXAN₆462, suggested that they maybe a gene cluster. We also demostrated that the promoter of MXAN₆461 itself were located on the upstream of MXAN₆462. What’s more, we identified that MXAN₆460 coded the ECF anti-σ factor.To sum up, we studied almost all the ECF σ factors in M. xanthus, trying to screen their roles in the growth, development, sporulation, and motility under differnt environment stress. Then, we analyed the transcription of MXAN₆461 and tried to find its promoter and genes that were regulated by MXAN₆461. Our work may offer an example for the research of other ECF σ factors in myxobacteria and contributed to discover some new mechanisms of ECF σ factors.