Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae

An analysis of previous data indicated that four structural genes concerned with maltosaccharide utilization in Streptococcus pneumoniae are organized in two operons that are transcribed in opposite directions from a central control region. This region contains two strong promoters subject to repression by a regulatory gene product in the absence of maltose. The nucleotide sequence of the 554-bp control region DNA and adjacent portions of the malX and malM structural genes was determined. Unique reading frames and initiation codons allowed identification of the oppositely oriented structural genes. Putative ribosome binding sites and −10 and −35 RNA-polymerase-binding sites, as well as AT-rich regions farther upstream, were observed proximal to both the X and M genes. The similarity of these sequences to sites found in Escherichia coli and Bacillus subtilis indicated the conservation of control signals in bacteria, both Gram-negative and Gram-positive. A pair of 17-bp hyphenated repeat sequences in the control region may represent repressor binding sites. Two down promoter mutations, V11 and 69, were shown to be deletions in the control region. The V11 mutation, which affected only the MP operon, deleted the promoter adjacent to the M gene. Mutation 69, which reduced both X and M gene functions, deleted the entire segment between the promoters so that they now overlap at their −35 binding sites. As a consequence of this deletion, the AT-rich regions proximal to the promoters were lost. This suggests that the AT-rich regions are important for promoter strength.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.     

Bacillussubtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-tenninal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.     

The tyrosyl-tRNA synthetase from Escherichia coli: Complete nucleotide sequence of the structural gene

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on M_r of 47 403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Non-periodic cues generate seven ftz stripes in the Drosophila embryo

We have examined the expression pattern of the segmentation gene fushi tarazu (ftz) by in situ hybridization to whole mount embryos using digoxygenin labeled probes. This method has revealed previously undetected stages in the development of the ftz RNA pattern. The ftz stripes arise individually in a distinct, non-linear order along the anterior-posterior axis of the embryo. In addition, the stripes develop differentially along the dorsal-ventral axis; most stripes emerge on the ventral side and then gradually spread dorsally until they surround the entire circumference of the embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy (h) stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the ftz stripes. The contribution of cis-acting regulatory elements to the ftz pattern was examined. The zebra and upstream elements interact to generate seven correctly positioned stripes at the end of cellularization. However, stripe establishement is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date. These observations suggest that the ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with previously identified ftz regulatory elements. This multi-level regulation provides a back-up system that ensures the development of seven stripes in the blastoderm.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.     

豆科作物间作燕麦对土壤固氮微生物丰度和群落结构的影响

利用荧光定量PCR和高通量测序的研究方法,比较了3种种植模式(燕麦单作,O;大豆/燕麦,OSO;绿豆/燕麦,OMO)对燕麦土壤固氮微生物数量和群落组成的影响.结果表明: 与大豆和绿豆间作显著改变了燕麦土壤的理化性质.燕麦土壤固氮微生物nifH基因拷贝数为每克干土1.75×10¹⁰～7.37×10¹⁰,拔节期和成熟期OSO和OMO中nifH基因拷贝数分别是O中的2.18、2.64和1.92、2.57倍,且各处理成熟期nifH基因拷贝数显著低于拔节期.样品稀释性曲线和覆盖度结果表明,各样品nifH基因测序结果可靠.与绿豆间作显著提高了燕麦土壤nifH基因的多样性.各样品固氮微生物属水平上优势类群均为Azohydromonas、固氮菌属、慢生根瘤菌属、Skermanella和在属水平上无法归类的固氮微生物,但各优势类群相对丰度存在差异.样品OTU的venn分布和主成分分析显示,拔节期和成熟期nifH基因群落结构存在差异,两个生育时期OSO和OMO具有更相似的nifH基因群落结构.表明与大豆和绿豆间作可显著提高燕麦土壤固氮微生物的数量,并影响固氮微生物的群落组成.     

Escherichia coli leucine-responsive regulatory protein (Lrp) controls lysyl-tRNA synthetase expression

Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase. The transposon giving rise to the altered expression of lysU was found inserted within lrp. The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine. An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion. The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.     

不同恢复方式对退化高寒草甸土壤nifH和chiA微生物群落结构的影响

生物固氮和有机氮降解是土壤有效氮的主要来源,固氮和有机氮降解微生物对土壤氮素供应和地力维持具有重要作用。本研究以青藏高原4种不同利用方式的高寒草甸(未扰动、放牧、围封和围封+补播草甸)为对象,结合荧光实时定量和扩增子测序技术,研究了不同恢复方式对nifH固氮菌和chiA几丁质降解菌基因丰度及其微生物群落结构的影响。结果表明: 4种草甸的nifH和chiA基因丰度大小排序为: 未扰动草甸＞放牧草甸＞围封草甸＞围封+补播草甸,其中未扰动草甸的nifH和chiA基因丰度分别是其他3种草甸的3.4～6.3倍和3.3～8.3倍;固氮菌α多样性在放牧、围封和围封+补播草甸中显著高于未扰动草甸,而几丁质降解菌的α多样性在未扰动和放牧草甸中最高;放牧显著提高了变形菌的丰度而降低了蓝细菌和放线菌丰度;土壤含水量、养分和植被特征对nifH和chiA的基因丰度和群落结构均有显著影响。与未扰动草甸相比,放牧降低了高寒草甸的固氮和有机氮降解潜力,而10年的围封禁牧和补播植草措施对固氮和有机氮降解功能的改善作用不明显。草甸恢复时应综合考虑功能微生物特征及其影响因素,放牧草甸恢复至未扰动水平可能需要更长的封育时间或者采用更加合理的管护措施。     

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an M_r of 56 668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tic revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited an M_r of approx. 34000.     

Cloning and expression of the Zymomonas mobilis pyruvate kinase gene in Escherichia coli

The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4 kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.     

Effect of sucA or sucC gene knockout on the metabolism in Escherichia coli based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic fluxes by C-labeling experiments

The effect of sucA or sucC gene knockout on the metabolism in Escherichia coli was investigated for the aerobic cell growth in batch and continuous cultivations based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic flux analysis. In the batch cultivation, the cell growth rate and the glucose uptake rate were lower for sucA mutant as compared with the parent strain, while it was not the case for sucC mutant. A significantly higher amount of acetate was produced, and it was not utilized in sucC mutant, while a little less acetate was produced in sucA mutant as compared with the parent strain. Unlike the parent strain and sucC mutant, sucA mutant excreted a little amount of l-glutamate. Enzyme activity results show that some of the glycolytic enzymes such as Tpi and Pgk were up-regulated, while Pfk, Fba and Pyk activities were down-regulated for sucA mutant as compared with the parent strain. For sucC mutant, the activities of Pfk, Fba, Tpi, GAPDH, Pgk and Pyk activities were down-regulated. As for the TCA cycle enzymes, the activities of CS and ICDH were down-regulated, while those of Icl, MS, Fum and MDH were up-regulated for sucA mutant. The activities of the oxidative pentose phosphate (PP) pathway enzymes such as G6PDH and 6PGDH and the gluconeogenic pathway enzyme such as Mez were up-regulated in sucA mutant. The Ack activity was down-regulated for sucA mutant, but not for sucC mutant. In continuous cultivation, the gene expression results indicate that the global regulatory genes such as fadR and iclR were slightly down-regulated in sucA mutant, which enhanced the expression of aceA gene and caused the up-regulation of the isocitrate lyase activity in sucA mutant, while fadR and iclR of sucC mutant changed little and no isocitrate lyase activation was observed for sucC mutant. Some other global regulatory genes such as arcA and fnr genes were down-regulated in both mutants, which caused some of the TCA cycle genes to be up-regulated. The effect of the sucA gene knockout on the metabolic flux distributions was investigated based on ¹H–¹³C NMR spectra and GC–MS signals obtained from ¹³C-labeling experiments. Flux analysis results indicate that the knockout of sucA gene caused the activation of PP pathway and the glyoxylate shunt. The fluxes through glycolysis and the TCA cycle were down-regulated in the sucA mutant. On the other hand, the fluxes through PP pathway and the anaplerotic reactions of Ppc-Pck and Mez increased.     

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with M_rs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.