Cloning and characterization of the DNA region responsible for Megacin A-216 production in Bacillus megaterium 216

Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.     

Sheep Wool δ13C Reveals No Effect of Grazing on the C3/C4 Ratio of Vegetation in the Inner Mongolia–Mongolia Border Region Grasslands

We tested whether the abundance of C₄ vegetation in grasslands of the Mongolian plateau is influenced by grazing conditions. The analysis exploited the politically originated contrast that exists between Mongolia (low stocking rate, transhumant system) and the district of Inner Mongolia, China (high stocking rate, sedentary system). We estimated the proportion of C₄ carbon (P_C4) in grazed vegetation from the relative carbon isotope ratio (δ¹³C) of sheep wool sampled from 298 annual shearings originating from 1996 to 2007. Annual stocking rates varying over time and between the districts of both countries were taken from regional statistics. The P_C4 pattern within the 0.7 million km² sampling area was geostatistically analyzed and related to stocking rates and temperature gradients. For similar climatic conditions, P_C4 was the same in both countries. Further, a unique relationship was found between P_C4 and July temperature on both sides of the border, which explained 71% of the pattern. Stocking rate and grazing system had no significant influences on present-day C₃/C₄ abundance ratio. This finding suggests that recent changes in the C₃/C₄ ratio of these grasslands are mainly a consequence of regional warming, not overgrazing.     

An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity

To test their structural and functional similarity, hybrids were constructed between EcoRI and RsrI, two restriction endonucleases recognizing the same DNA sequence and sharing 50% amino acid sequence identity. One of the chimeric proteins (EERE), in which the EcoRI segment His147-Ala206 was replaced with the corresponding RsrI segment, showed EcoRI/RsrI-specific endonuclease activity. EERE purified from inclusion bodies was found to have approximately 100-fold weaker activity but higher specific DNA binding affinity, than EcoRI. Increased binding is consistent with results of molecular dynamics simulations, which indicate that the number of hydrogen bonds formed with the recognition sequence increased in the chimera as compared to EcoRI. The success of obtaining an EcoRI-RsrI hybrid endonuclease, which differs from EcoRI by 22 RsrI-specific amino acid substitutions and still preserves canonical cleavage specificity, is a sign of structural and functional similarity shared by the parental enzymes. This conclusion is also supported by computational studies, which indicate that construction of the EERE chimera did not induce substantial changes in the structure of EcoRI. Surprisingly, the chimeric endonuclease was more toxic to cells not protected by EcoRI methyltransferase, than the parental EcoRI mutant. Molecular modelling revealed structural alterations, which are likely to impede coupling between substrate recognition and cleavage and suggest a possible explanation for the toxic phenotype.     

Yersinia pestis lineages in Mongolia

Background

Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia.

Methodology/Principal Findings

Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches.

Conclusions/Significance

We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.     

耐受微酸性次氯酸水乳酸菌的分离鉴定、耐受特性及益生特性

【背景】次氯酸水具有杀菌性,当其作为饮用水可能会对畜禽肠道内有益菌造成负面影响。【目的】筛选出具有耐受微酸性次氯酸水(slightly acidic hypochlorous acid water, SAHW)能力的乳酸菌并研究其益生特性。【方法】从内蒙古地区传统自然发酵制品中分离筛选出8株乳酸菌菌株,经16SrRNA基因测序进行鉴定。进一步通过耐受SAHW、耐酸、耐胆盐、疏水性及自凝聚能力试验进行耐受特性和益生特性研究。【结果】菌株MBH3-2为副干酪乳杆菌,其经过质量浓度为20mg/L的SAHW处理2h,菌落对数值仅下降0.3lg(CFU/mL),存活率为50.10%;而且在酸性(pH 2.0、2.5和3.0)、胆盐环境下均可存活;疏水率达62.12%,属于高度疏水性;自凝聚能力在2 h达到97.29%。【结论】最终确定一株具有优良耐受SAHW及基本满足作为微生态制剂要求的菌株,MBH3-2具有作为新型微生态制剂菌株的潜力。