| As remarked by the Nobel Laureate Francois Jacob,"The dream of every cell is to become two cells".Rapid growth and proliferation is one of the core properties of bacteria.In nature,the living environment varies greatly among different bacterial species.During the long evolutionary process in adapting to various environments,there exist a huge difference in the growth capacity of different bacteria species.The maximal growth rates of different bacteria range from 15 minutes per generation to several days per generation.The control of bacteria growth is one of the fundamental topics in microbiology.However,previous studies on bacterial growth control mainly focus on the fast-growing model bacterium Escherichia coli,and little knowledge is known about slow-growing bacterial species.Mycobacterium smegmatis is a kind of slow-growing soil bacteria with its maximal growth rate being no faster than 3 hours per generation.Currently,little is known on its basic growth and physiological proprieties.In the first part of my thesis,I systematically studied the effects of various environmental factors such as nutrient quality,osmolarity,high-and low-temperature,and ethanol treatment on the growth physiology of M.smegmatis.In the second part of the thesis,I focused on the(p)ppGpp synthetase of M.smegmatis.Previous studies have shown that guanosine tetra/pentaphosphate,(p)ppGpp is a key global regulator of the growth of E.coli.In my thesis,I studied the functional complementation of the(p)ppGpp synthetase gene(relMsm)of M.smegmatis in E.coli.In the third part of the thesis,I established an inducible expression vector in M.smegmatis.In the first part of the thesis,I systematically studied the effects of various environmental factors on the growth of M.smegmatis,including nutrient quality,temperature,ethanol treatment,osmolarity and several commonly used antibiotics.I prepared various nutrient conditions through using different combinations of carbon sources and nitrogen sources.In these conditions,the generation time of M.smegmatis range from 150 min to 700 min.I found that different carbon and nitrogen sources could significantly affect the growth rate of M.smegmatis.For example,glycerol and succinate are good carbons that could support relatively fast growth while sorbitol is the worst carbon source in my study.Moreover,ammonium is a good nitrogen source while nitrate and nitrite are poor nitrogen sources.It was further found that the ribosome content of M.smegmatis was positively correlated with the growth rate under different nutrient conditions,that is,the faster the cell growth,the higher the ribosome content.The lacZ reporter gene system was used to determine the promoter activity of the two ribosomal RNA(rRNA)operons of M.smegmatis,rrnA and rrnB.The transcription activity of rrnA operon is significantly higher than that of rrnB.However,in contrast to the pattern of cellular ribosome content,the promoter activities of both rrnA and rrnB are negatively correlated with the growth rate,suggesting that positive growth rate-dependence of ribosome content under different nutrient conditions does not originate from the regulation of transcriptional activity of the rrn promoter.Several other environmental factors could also significantly affect the growth of M.smegmatis:1)Temperature;The optimal growth temperature of M.smegmatis is 37℃ to 45℃,and both low temperature(such as 25℃)and high temperature(48℃)inhibit cell growth.However,ribosome content even decreases under low temperatures,and are thus not related to the growth inhibition by low temperatures.2)Ethanol;2%-4%ethanol could significantly reduce the bacterial growth rate,accompanied with a negative effect of the cellular ribosome content as well.3)Oxidative stress;when treated with a low level of hydrogen peroxide(0-4 mM)to medium,the growth of M.smegmatis was immediately arrested,and however,recovered after a certain lag period.The length of the lag period is positively correlated with the level of hydrogen peroxide.However,when the hydrogen peroxide concentration exceeds 5 mM,the growth of bacteria was completely inhibited and cell viability lost later.4)High osmolarity;0.2-0.8 M of sodium chloride(NaCl)could significantly reduce the growth rate of M.smegmatis but with no effect on the cellular ribosome content.In special,bacteria growing in good nutrient could tolerate a higher concentration of sodium chloride stress than their counterparts growing under poor nutrition.5)Antibiotic stress;0-10 μg/ml chloramphenicol,0-2μg/ml kanamycin,and 0-2 μg/ml norfloxacin are sublethal,causing reduction but not complete cessation of the growth of M.smegmatis.The second part of the paper,I studied the functional complementation of the(p)ppGpp synthetase gene(re1Msm)of M.smegmatis in E.coli.Previous studies have shown that guanosine tetra/pentaphosphate(p)ppGpp is a key global regulator of the growth of E.coli.There are two related genes for the metabolism of(p)ppGpp,relA(encoding a single-function synthetase)and spoT(encoding a(p)ppGpp synthesis/hydrolysis bi-function enzyme).Knockout of relA alone does not affect the growth of E.coli,but the knockout of spoT is lethal for E.coli.The ΔrelA ΔspoT double mutant strain,although could be made,but could not grow in minimal media.In the genome of M.smegmatis,there is only one homologous gene,relMsm.Although sequence alignment revealed that the RelMsm protein has only a 30%homology with the RelA and SpoT proteins of E.coli.The expression vector of relMsm gene in E.coli could allow the knockout of the spoT gene of E.coli and could allow the ΔrelAΔspoT double knockout bacteria to grow in minimal medium,confirming that the relMsm has a similar function with spoT gene and encodes a(p)ppGpp synthesis/hydrolysis dual function enzyme.In the last part of the thesis,I established an inducible expression system of M.smegmatis.I cloned the promoter PamiE(The promoter of the acetamidase gene in M.smegmatis)and its upstream regulatory element into a pAL5000 replicon vector and fused the lacZ reporter gene with PamiE.By supplementing different concentrations of acetamides in the medium,I found that the activity of the report system could be induced by 15-fold and no effect on the bacteria growth rate,indicating that this system is a well inducible system.In the future,it could be used to titrate the expression of related genes to investigate their functions.In summary,my thesis systematically studies the effect of various environmental factors on the growth and physiology of M.smegmatis,and have confirmed that its relMsm gene,being similar to the spoT gene of E.coli,encoding a(p)ppGpp synthesis/hydrolysis double function enzyme.Finally,the thesis has also established an inducible expression system for the M.smegmatis.My research is expected to lay a good foundation for future investigation of the growth control of M.smegmatis. |
PDF Full Text Request