Characterization Of PucBA Deletion Mutants From Rhodopseudomonas Palustris

As a group of anaerobic phototrophic bacteria,purple bacteria have evolved a number of responses that enable them to adapt to the variable light environment,which have served as the important models for the study of photosynthesis mechanisms.Besides changes in the amount of light-harvesting complex 2?LH2,B800-850?,some species of purple bacteria have an additional response,producing the abnormal absorption spectra of LH2,such as LH3?B800-820?or LH4?B800-low-850?under low light?LL?intensities,which was related to the replacement of the key residues on the?-apoprotein of LH2 and the multigene family of pucBA genes encoding LH2 complex?/?-polypeptides.Whether these LH2 with abnormal spectra are beneficial to the growth of bacteria under LL intensity has been proved in a few strains that LH3 has higher efficiency of energy transfer?EET?than LH2.However,in this study,it has been found that the EET of LH4 was lower than LH2 in Rhodopseudononas?Rps.?palustris CGA009,then the question was raised whether the synthesis of LH4 can promote the growth of bacteria under LL condition.Moreover,the details of LH2 complexes of which each ring is homogeneous or heterogeneous with individual rings having?/?apoproteins present that are encoded by different pucBA genes were still unclear.To address these issues,Rps.palustris CGA009 was used in our study which can synthesize LH4 under LL and five pairs of pucBA genes were scattered in its genome.The pucBAd gene in the genome of strain CGA009 was deleted to create the mutant without LH4 under LL,whose growth characteristics and the synthesis of photosynthetic apparatus were studied,providing direct evidence for the growth-promoting of LH4 to LL.And a new mechanism of light adaptability of LH4 was proposed by comparing the energy transfer characteristics of LH2 and LH4.Furthermore,four mutant strains?pucBA_da,?pucBA_dab,?pucBA_dabeabe and?pucBA were obtained by knocking out the other pucBA genes in strain CGA009 one by one in order to explore the effects of multiple pucBA on the photosynthetic characteristics of the strain.A genetic system for the complementary expression of pucBA gene was attempted to provide new models for the further study on the interaction between?/?peptides encoded by different pucBA genes and the assembly of LH2.The main results obtained were as follows.Firstly,the mutant?pucBA_d was obtained by knocking out the gene encoding LH4?/?peptide in strain CGA009.The characteristics of mutant and wild-type?WT?strain were compared,including growth rate,absorption spectra of living cells and photosynthetic membranes,content and composition of photosynthetic pigments?BChl a and Car?.Under high light?HL?intensity,the characteristics of mutant and WT strain were not significantly different,yet under LL intensity,the growth rate of mutant without the characteristic absorption peak of LH4 decreased markedly,and its content of photosynthetic pigments was not strikingly different from that of WT strain,indicating that the deletion of LH4 did not affect the growth of strain CGA009under HL,but was bad for the growth of strain CGA009 under LL.The fluorescence spectra of LH2 and LH4 was compared and the results were obtained that the fluorescence peak of LH2 and LH4 was about at 860 and 850 nm,respectively.Compared with LH2,the fluorescence peaks of LH4 was blue-shifted and its peak area was larger.A new mechanism was proposed to explain the adaptability of LH4under LL.Compared with LH2 which has a strong absorption peak of 850 nm,LH4absorbs little fluorescence at the same position,indicating that LH4 can provide more photons with higher energy,and thus has higher energy transfer activity.Secondly,on the basis of mutant?pucBA_d,the other four pucBA genes were knocked out one by one,creating four mutant strains(?pucBA_da,?pucBA_dab,?pucBA_dabeabe and?pucBA)which were confirmed by gene analysis and absorption spectra of living cells.Compared with the spectral characteristics of LH2 and LH4synthesized by strain CGA009,the Q_y bands of mutants?pucBA at high and low light intensity were 804 and 871 nm,804 and 875 nm,respectively,suggesting that the quintuple mutants no longer synthesized LH2 and only contained RC-LH1.The growth rate of?pucBA was slower and its content of photosynthetic pigments decreased under HL and LL,showing that the light-harvesting efficiency of?pucBA without LH2 was reduced.Finally,we cloned pucBAd encoding the?/?peptide of LH4 to the expression vector?pBBR1MCS-2 and pVLT33?which was transferred into?pucBA,in order to establish a genetic system for the complementary expression of pucBA genes.The characteristic absorption spectra of high-expressed of LH4 was not detected by living cell absorption spectroscopy,indicating that the expression and assembly of LH4 were not obvious.In summary,this study provides experimental evidence for the adaptability of LH4 to LL that the synthesis of LH4 under LL is beneficial to the growth of strain CGA009,and a new mechanism for the growth-promoting of LH4 under LL was proposed.The strain CGA009 with all deletion of multiple of pucBA was obtained by gene knockout,providing an ideal strain for the research of interactions between?/?peptide encoded by multicopies pucBA gene.We tried the expression of pucBAd gene in?pucBA,improving the genetic system of anaerobic phototrophic bacteria.Further research is needed on genetic tools for the interaction between?/?peptides encoded by different pucBA genes and the correct assembly of LH2.