Running Title Evolution of the LexA binding sequence
An article to Microbiology
Section: Genes and Genomes
Reconstruction of the evolutionary history of the LexA binding sequence
by
Gerard Mazón1, Ivan Erill2, Susana Campoy3, Pilar Cortés1, Evelyne Forano4 and Jordi Barbé 1,3,5
1 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona 08193 Bellaterra, Spain
2 Biomedical Applications Group, Centro Nacional de Microelectrónica, 0819
3 Bellaterra, Spain
3 Centre de Recerca en Sanitat Animal (CReSA), 08193 Bellaterra, Spain
4Unité de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France
5 To whom reprint requests should be addressed.
E-mail: jordi.barbe@uab.es
Phone: 34 - 93 – 581 1837
Fax: 34 – 93 – 581 2387
Running Title: Evolution of the LexA binding sequence
Keywords: lexA, SOS response, evolution, lateral gene transfer
SUMMARY
In recent years, the recognition sequence of the SOS repressor LexA protein has been identified for several bacterial clades, such as the Gram-positive, Green-non Sulfur bacteria and Cyanobacteria phyla, or the Alpha, Delta and Gamma Proteobacteria classes. Nevertheless, the evolutionary relationship among these sequences and the proteins that recognize them has not been analyzed. Fibrobacter succinogenes is an anaerobic Gram-negative bacterium that branched from a common bacterial ancestor immediately before the Proteobacteria phylum. Taking advantage of its intermediate position in the phylogenetic tree, and in an effort to reconstruct the evolutionary history of LexA binding sequences, the F. succinogenes lexA gene has been isolated and its product purified to identify its DNA recognition motif through electrophoretic mobility assays and footprinting experiments. After comparing the available LexA DNA binding sequences with the here reported F. succinogenes one, directed mutagenesis of the F. succinogenes LexA binding sequence and phylogenetic analyses of LexA proteins have revealed the existence of two independent evolutionary lanes for the LexA recognition motif that emerged from the Gram-positive box: one generating the Cyanobacteria and Alpha Proteobacteria LexA binding sequences, and the other giving rise to the F. succinogenes and Myxococcus xanthus ones, in a transitional step towards the current Gamma Proteobacteria LexA box. The contrast between the results here reported and the phylogenetic data available in the literature suggests that, some time after their emergence as a distinct bacterial class, the Alpha Proteobacteria lost its vertically received lexA gene, but received later through lateral gene transfer a new lexA gene belonging to either a Cyanobacterium or a bacterial species closely related to this Phylum. This constitutes
the first report based on experimental evidence of lateral gene transfer in the evolution of a gene governing such a complex regulatory network as the bacterial SOS system.
INTRODUCTION
Preservation of genetic material is one of the most fundamental functions of any living being and it is perhaps in the Bacteria Domain where this aspect has been most thoroughly studied. As in the case of many other biological processes, Escherichia coli has been the principal subject of this research, and many E. coli genes involved in preservation of genetic material have been identified through the years. Some of them encode proteins that are able to repair different types of DNA injuries, whilst others aim at guaranteeing cell survival in the presence of such lesions. Many of these genes act in a coordinate manner, constituting specific DNA repair networks, and the broadest and most thoroughly studied of these regulons is the LexA-mediated SOS response (Walker, 1984). In E. coli, the LexA protein controls the expression of some 40 genes (Fernandez de Henestrosa et al., 2000; Courcelle et al., 2001), including both the lexA and recA genes, which are, respectively, the negative and positive regulators of the SOS response (Walker, 1984). The E. coli LexA protein specifically recognizes and binds to an imperfect 16-bp palindrome with consensus sequence CTGTN8ACAG, designated as the E. coli SOS or LexA box (Walker, 1984). Both in vitro and in vivo experiments have shown that binding to single-stranded DNA fragments generated by DNA damage-mediated inhibition of replication activates the RecA protein (Sassanfar & Roberts, 1990). Once in its active state, RecA promotes the autocatalytic cleavage of LexA, resulting in the expression of the genes regulated by this repressor (Little, 1991). Hydrolysis of the E. coli LexA protein is mediated by its Ser119 and Lys156 residues, in a mechanism similar to that of proteolysis by serine proteases (Luo et al., 2001). After DNA repair, the RecA protein ceases to be
activated and, consequently, non-cleaved LexA protein returns to its usual levels, repressing again the genes that are under its direct negative control.
Even though some notable exceptions have been reported, the increasing availability of microbial genome sequences has revealed that LexA is present in many bacterial species and in most phyla. So far, all the identified and characterized LexA proteins display two conserved domains that are clearly differentiated. The N-domain, ending at the Ala-Gly bond where the protein is cleaved after DNA damage activation of RecA (Little, 1991), has three α helices that are necessary for the recognition and binding of LexA to the SOS box (Fogh et al., 1994; Knegtel et al., 1995). Conversely, the C-domain contains amino acids that are essential for the serine-protease mediated auto-cleavage and for the dimerization process necessary for repression (Luo et al., 2001).
The sequence of the LexA box is strongly conserved among related bacterial species. In fact, the LexA box has been shown to be monophyletic for several bacterial phyla, and this feature has been successfully exploited in phylogenetic analyses (Erill et al., 2003). Thus, in the Gram-positive Phylum the LexA binding motif presents a CGAACRNRYGTTYC consensus sequence (Winterling et al., 1998) that, with slight variations (Davis et al., 2002), is conserved among all its members and is also found in the phylogenetically close Green Non-sulfur Bacteria that, nonetheless, are Gram-negative bacteria (Fernandez de Henestrosa et al., 2002). Apart from the Gamma Proteobacteria, in which the consensus sequence CTGTN8ACAG is monophyletic and seems to extend to those Beta Proteobacteria that present a lexA gene (Erill et al., 2003), alternative LexA binding sequences with a high degree of conservation have also been described in other groups. So far, for instance, the direct repeat GTTCN7GTTC is the LexA binding sequence of the Alpha Proteobacteria harboring a
lexA gene, a group that includes the Rhodobacter, Shinorizobium, Agrobacteium, Caulobacter and Brucella genera (Fernandez de Henestrosa et al., 1998; Tapias & Barbe, 1999). Still, in other phyla where the LexA binding motif has been identified more data is required to gauge the conservation of the LexA box. Such is the case of the Delta Proteobacteria, for which a CTRHAMRYBYGTTCAGS consensus motif has been identified in one of its members, the fruiting body forming Myxococcus xanthus (Campoy et al., 2003).
The existence of different LexA recognition motifs and the monophyletic or paraphyletic nature of those studied so far indicate that the appearance of new LexA binding motifs marks turning points in the evolutionary history of both this protein and its respective host species. Previous work has demonstrated that the Cyanobacteria LexA box (RGTACNNNDGTWCB) derives directly from that of Gram-positive bacteria (Mazón et al., 2004). Nevertheless, a huge gap is still apparent in the further evolutionary pathway of the LexA box that leads from the Cyanobacteria up to other bacterial phyla of later appearance, such as the Proteobacteria. Protein signature analyses have established that Fibrobacter succinogenes branched from a common bacterial ancestor immediately before the Proteobacteria phylum (Griffiths & Gupta, 2001). F. succinogenes is an anaerobic gram-negative bacterium that inhabits the rumen and caecum of herbivores and, for a long time, this organism was included in the Bacteroides genus. Recent 16S rRNA analyses, however, have challenged this notion, and Fibrobacter has been granted a new Bacterial Phylum of its own (Maidak et al., 1999; Ludwig & Schleifer, 1999).
In an effort to recreate the evolutionary history of the LexA protein through the changes in its recognition sequence, and taking advantage of the fact that the F. succinogenes genome is now partially sequenced, the lexA gene of this bacterial
species has been isolated and its encoded product has been purified to determine its DNA recognition sequence. The results here obtained are in accordance with the newly established branching point of F. succinogenes, and introduce a novel element that allows a finer drawing of the evolutionary path of the LexA recognition sequence from Gram-positive bacteria to Gamma Proteobacteria.
METHODS
Bacterial strains, plasmids, oligonucleotides and DNA techniques. Bacterial strains and plasmids used in this work are listed in Table 1. E. coli and F. succinogenes ATCC19169strains were grown at either in LB (Sambrook et al., 1992) or in a chemically defined medium (Gaudet et al., 1992) with 3 g l-1 of cellobiose, respectively. Antibiotics were added to the cultures at reported concentrations (Sambrook et al., 1992) . E. coli cells were transformed with plasmid DNA as described (Sambrook et al., 1992). All restriction enzymes, PCR-oligonucleotide primers, T4 DNA ligase and polymerase, and the "DIG-DNA labelling and detection kit" were from Roche. DNA from F. succinogenes cells was extracted as described (Forano et al., 1994).
The synthetic oligonucleotide primers used for PCR amplification are listed in Table 2. To facilitate subcloning of some PCR-DNA fragments, specific restriction sites were incorporated into the oligonucleotide primers. These restriction sites are identified in Table 2. Mutants in the F. succinogenes lexA promoter were obtained by PCR-mutagenesis, using oligonucleotides carrying designed substitutions (Table 2). The DNA sequence of all PCR-mutagenized fragments was determined by the dideoxy method (Sanger et al., 1977) on an ALF Sequencer (Amersham-Pharmacia). In all cases the entire nucleotide sequence was determined for both DNA strands.
Molecular cloning of the F. succinogenes lexA gene and purification of its encoded protein. The F. succinogenes lexA gene was amplified from the total DNA of the F. succinogenes ATCC19169 strain using the LexAup and LexAdwn oligonucleotide primers (Table 2) corresponding to nucleotides –276 to –249 and +653 to +678, with respect to its proposed translational starting point. The 954-bp PCR fragment obtained was cloned into the pGEM-T vector (Promega) obtaining the pUA1033 plasmid. To confirm that no mutation was introduced during the amplification reaction, the sequence of the fragment was determined. The plasmid pUA1038 was constructed in order to create and express a Glutathione-S-transferase (GST)- F. succinogenes LexA fusion protein. The first step in the construction of this plasmid was to amplify the F. succinogenes lexA gene from plasmid pUA1033, using the primers LexAEcoRI and LexADw. The resulting DNA fragment was cloned into pGEM-T, to give rise to pUA1037. Following excision with Eco RI and Sal I, the lexA gene was inserted into the pGEX4T1 expression vector (Amersham-Pharmacia), immediately downstream of the GST-encoding gene that is under the T7 promoter control. The initiation codon of the LexA protein was placed immediately downstream of the Eco RI sites in LexAEcoRI primer, such that the lexA gene could be fused to GST in frame. The insert of pUA1037 was sequenced in order to ensure that no mutations were introduced during amplification.
To overproduce the LexA-GST fusion protein, the pUA1037 plasmid was transformed into E. coli BL21(λDE3) codon plus strain (Stratagene). Cells of the resulting BL21 codon plus strain were diluted in 0.5 L of LB medium and incubated at 37o C until they reached an O.D.600 of 0.8. Fusion protein expression was induced at this time by the addition of IPTG to a final concentration of 1 mM. Following incubation for an additional 3 h at 37o C, cells were collected by centrifugation for 15
min at 3 000 g. The bacterial pellet was resuspended in PBS buffer (10 mM Na2HPO4, 1.7 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl -pH 7.4-), containing ?Complete Mini? protease inhibitors cocktail (Roche). The resulting cell suspensions were lysed by sonication. Unbroken cells and debris were removed by centrifugation for 20 min at 14 000 g. The supernatant containing the GST-LexA fusion protein was incubated with PBS-Glutathione Sepharose 4B? beads (Amersham Pharmacia), for 2 h at 4o C, in order to affinity purify the fusion protein. The beads were then washed twice with PBS containing 0.1% Triton and three times with PBS without detergent. The sequence Leu-Val-Pro-Arg-Gly-Ser is located immediately downstream of the GST coding sequence in the pGEX4T vector series, and serves as a linker between the LexA and GST moieties of the fusion proteins. This hexapeptide is recognized by the protease thrombin, which cleaves at the Arg-Gly bond. It was therefore possible to release the F. succinogens LexA protein from the sepharose beads by incubating a 700 μl bed volume of beads with 25 Units of thrombin (Amersham - Pharmacia) in 1 ml of PBS. The supernatants containing the F. succinogenes LexA protein with an additional five amino acid tail at their N-terminal (Gly-Ser-Pro-Glu-Phe), was visualized in a Coomassie blue stained 13% SDS-PAGE gel (Laemmli, 1970). Their purity was greater than 98% (data not shown).
LexA proteins from B. subtilis, E. coli, Anabaena PCC7120, M. xanthus and R. sphaeroides also used in this work had been previously purified (Winterling et al., 1998; Tapias et al., 2002; Campoy et al., 2003; Mazón et al., 2004).
Mobility shift assays and DNase I footprinting. LexA-DNA complexes were detected by electrophoresis mobility shift assays (EMSAs) using purified LexA proteins. DNA probes were prepared by PCR amplification using one of the primers labelled at its 5’ end with digoxigenin (DIG) (Table 2), purifying each product in a
2% -3% low-melting-point agarose gel depending on DNA size. DNA-protein reactions (20 μl) typically containing 10 ng of DIG-DNA-labelled probe and 40 nM of the desired purified LexA protein were incubated in binding buffer: 10 mM N-2-Hydroxyethyl-piperazine-N’ 2-ethanesulphonic acid (HEPES) NaOH (pH 8), 10 mM Tris-HCl (pH 8), 5% glycerol, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 2 μg poly(dG-dC) and 50 μg/ml of BSA. After 30 minutes at 30°C, the mixture was loaded onto a 5% non-denaturing Tris-glycine polyacrylamide gel (pre-run for 30 minutes at 10 V/cm in 25mM Tris-HCl (pH 8.5), 250mM glycine, 1mM EDTA). DNA-protein complexes were separated at 150 V for 1 hr, followed by transfer to a Biodine B nylon membrane (Pall Gelman Laboratory). DIG-labelled DNA-protein complexes were detected by following the manufacturer’s protocol (Roche). For the binding-competition experiments, a 300-fold molar excess of either specific or unspecific-unlabelled competitor DNA was also included in the mixture. Protein concentrations were determined as described (Bradford, 1976). All EMSAs were repeated a minimum of three times to ensure reproducibility of the results.
DNase I footprinting assays were performed using the ALF Sequencer (Amersham Biosciences) as described previously (Patzer and Hantke 2001; Campoy et al., 2003). In silico phylogenetic analysis. Preliminary sequence data of F. succinogenes unfinished genome was obtained from The Institute for Genomic Research (TIGR) through their website at https://www.wendangku.net/doc/2f15499681.html,, and protein sequences for all other organisms were obtained from the Microbial Genome Database for Comparative Analysis website (http://mbgd.genome.ad.jp/) and the TIGR Comprehensive Microbial Resource (CMR). Identification of additional LexA-binding genes was carried out using the RCGScanner software (Erill et al., 2003), using known E. coli LexA-governed genes (Fernández de Henestrosa et al., 2000; Erill et al., 2003) and
the here reported LexA box of F. succinogenes to scan and then filter through the consensus method putative LexA binding sites across the F. succinogenes genome. For phylogenetic analyses, protein sequences for each gene under study were aligned using the CLUSTALW program (Higgins et al., 1994). Multiple alignments were then used to infer phylogenetic trees with the SEQBOOT, PROML and CONSENSE programs of the Phylip 3.6 software package (Felsenstein, 1989), applying the maximum-likelihood method on 100 bootstrap replicates. The resulting phylogeny trees were plotted using TreeView (Page, 1996).
RESULTS
Determination of the F. succinogenes LexA recognition DNA sequence Electrophoretic mobility shift assays (EMSA) with the purified F. succinogenes LexA protein were carried out to determine the binding ability of this protein to its own promoter. As it can be seen in Fig. 1a, the addition of increasing concentrations of LexA to a fragment extending from –154 to +169 of the F. succinogenes lexA gene promoter (with respect to its proposed translational starting point) produces one retardation band whose intensity is directly related to the amount of protein used. The formation of this DNA-LexA complex is specific, since it is sensitive to competition by an excess of unlabelled lexA promoter, but not to competition by non-specific DNA (Fig. 1b). Moreover, EMSAs performed using different sized-fragments containing the lexA promoter as a probe demonstrated that the LexA recognition sequence must lie in a region included between positions –72 and –57 of this promoter (data not shown).
To precisely identify the F. succinogenes LexA box, additional footprinting experiments with a 160-bp fragment extending from positions –154 to +6 were
performed. The results obtained show that a 37 bp core region was protected by the LexA protein when both lexA-coding and non-coding strands were analyzed (Fig. 2).
A visual inspection of this DNA sequence revealed the presence of the imperfect palindrome TGCCCAGTTGTGCA in its central region. To determine whether this motif was really involved in LexA binding, the effect of single substitutions in each nucleotide of this palindrome on the formation of the LexA protein-lexA promoter complex was analyzed. Results (Fig. 3) indicate that a single substitution in any position of the TGC tri-nucleotide, as well as in the last C of the TGCCC motif, abolishes LexA binding. Likewise, mutagenesis of any position of the GTGCA motif does also inhibit DNA-LexA complex formation. On the contrary, the single substitution of nucleotides immediately surrounding either the TGCCC or the GTGCAT motifs does not affect LexA binding. Taken together, these results demonstrate that the TGCNCNNNNGTGCA imperfect palindrome is the LexA box of F. succinogenes, since it is required for the binding of this organism LexA protein to its own lexA promoter.
Identification of additional LexA-binding F. succinogenes genes
The characteristic amino acid residues of LexA proteins (an Ala-Gly bond separated about 34 positions from a Ser residue that is 37 positions away from a Lys residue) are also present, at least, in two other prokaryotic protein families: UmuD (encoding DNA polymerase V which is involved in error-prone DNA repair) and lytic cycle prophage repressors (such as the λcI protein) (Little, 1984; Burckhardt et al.,1988; Nohmi et al., 1988). Nevertheless, of these two proteins only the prophage repressors are able to bind DNA specific sequences. To discard the possibility that the F. succinogenes LexA was, in fact, a residual prophage repressor, an in silico analysis of
the F. succinogenes genome sequence was carried out using the RCGScanner program (Erill et al., 2003) in search of other genes with significant TGCNCNNNNGTGCA-like palindrome motifs upstream of their coding regions. The imperfect palindrome motif was found upstream the recA, uvrA, ssb and ruvAB genes, and competitive EMSA experiments demonstrated that their promoters also bind F. succinogenes LexA (Fig. 4). As it is known, these genes are under control of the LexA protein in many bacterial species and, therefore, the possibility that the F. succinogenes lexA gene here identified was a residual prophage repressor was discarded.
Comparative analysis of the F. succinogenes LexA protein and its recognition sequence
A sequence comparison between the aforementioned LexA-binding sequences and that of F. succinogenes reveals the presence of marked resemblances among several nucleotide positions (Fig. 5). On close inspection, this resemblance does not only suggest a common phylogenetic origin for these LexA proteins, but does also point at two putative evolutionary lanes emerging from the Gram-positive LexA box: one giving rise to the Cyanobacteria and Alpha Proteobacteria LexA box and the other leading to both F. succinogenes and M. xanthus in an intermediate step, and ultimately resulting in the Beta and Gamma Proteobacteria LexA box.
To validate this proposed separate branching in terms of LexA boxes, a phylogenetic tree (Fig. 6) was constructed from a multiple alignment of available LexA protein sequences from relevant members of the Gram-positive and Cyanobacteria phyla and the Alpha, Beta and Gamma Proteobacteria classes, and those of both F. succinogenes and M. xanthus. The resulting tree is clearly in accordance with the dual branching
hypothesis prompted by LexA boxes. Taking, as all available phylogenetic analyses suggest (Gupta & Griffiths, 2002), the Gram-positive bacteria as the closest Phylum to the common ancestor, two different LexA clusters emerge from that of this group (Fig. 6): the Cyanobacteria and Alpha Proteobacteria LexA protein cluster, and the Fibrobacter, Myxococcus and Beta and Gamma Proteobacteria LexA protein cluster.
Genesis of different LexA boxes through directed mutagenesis of the F. succinogenes LexA binding sequence
To further confirm the putative relationship between the LexA proteins described above, the vertical evolutionary path leading from Gram-positive bacteria to Gamma Proteobacteria was experimentally analyzed taking F. succinogenes LexA recognition sequence as a starting point to generate, through directed mutagenesis, the LexA binding sequences of Gram-positive, Myxococcus, Beta and Gamma Proteobacteria. As it can be seen (Fig. 7a), only five substitutions in the F. succinogenes LexA box (the T as well as the two internal Cs of the TGCCC motif, plus, the internal G and A of the GTGCAT one) are necessary to habilitate binding of the B.subtilis LexA protein to this F. succinogenes mutant promoter. Similarly, the M. xanthus LexA protein is able to bind a F. succinogenes lexA-derivative promoter in which only the flanking bases at both ends of the TGCCCAGTTGTGCA palindrome have been substituted for a C and a G, respectively, and the T of the internal GTGCAT motif has been replaced by a C. Finally, the E. coli LexA protein can effectively bind to the lexA promoter recognized by the M. xanthus LexA if only three additional changes to the mutant promoter are made: substitution of the CC duet for TA on the TGCC tetra-nucleotide and a change from T to A in the TTC tri-nucleotide.
Derivation of the Alpha LexA binding sequence from the Cyanobacterial LexA box
To complete the above described analysis on the evolutionary relationship of LexA proteins through their binding sequences, a similar study was conducted to check the feasibility of the remaining branching line from Gram-positive bacteria (i.e. the one giving rise to Cyanobacteria and Alpha Proteobacteria LexA proteins). In concordance with the hypothesis presented in Fig. 5, it was found that the simple addition of three nucleotides (chosen in accordance with the Alpha LexA-box consensus sequence) between the AGTAC and GTTC motifs of the Cyanobacterial LexA box was sufficient to enable the binding of the R. sphaeroides LexA protein to this mutant LexA box in the Anabaena lexA gene promoter (Fig. 7b). Furthermore, and although significant binding of the R. sphaeroides LexA protein to the Anabaena lexA promoter could be easily accomplished with the single insertion event described above, the introduction of an additional single-point mutation (substitution of T for A in the GTAC tetra-nucleotide) to the mutant Anabaena lexA promoter dramatically increased the recognition ability of the R. sphaeroides LexA repressor (Fig. 7b).
DISCUSSION
In this work we have demonstrated that, through a programmed set of nucleotide changes, both the Gram-positive and E. coli-like LexA boxes can be obtained from the F. succinogenes LexA binding sequence. Furthermore, our results point out that the G and C corresponding to the most external positions of the GAACN4GTTC motif recognized by the Gram-positive LexA repressor are enclosed in the CTGT and ACAG sequences, respectively, found in the E. coli-like LexA box. In this way, the origin of the E. coli LexA recognition sequence (constituted by 16 nucleotides) could
be explained by a 2-bp size increase of the Gram-positive LexA binding sequence (12 nucleotides long) through each one of its ends. Nevertheless, this extension of the LexA recognition motif does not seem to have carried a significant increase in the size of the N-domain region of the LexA protein that contains the three α helices involved in DNA binding (Fig. 8). A straight comparison of the N-terminal domain of F. succinogenes and M. xanthus LexA protein sequences with the consensus sequences of this region of Gram-positive, Cyanobacteria and Alpha Proteobacteria LexA proteins reveals no amino acid insertions in those residues that, in E. coli, have been shown to participate directly in DNA binding activity, nor in their immediate neighbors (Fig. 8).
Moreover, this comparative analysis of LexA protein sequences shows several fully conserved residues amongst those that constitute the three predicted α helices that are involved in DNA-binding. This suggests that, since their respective LexA boxes are markedly different, these amino acids must be required for the maintenance of the overall DNA recognition complex instead of being used for specific binding. This is the case for T5, Q8, E10, P26, S39, L50, G54 and R64, following the numeric position in the E. coli LexA protein. Likewise, other residues present a low degree of substitutions that, besides, correspond to amino acids of the same family: L4, I15, E30, L47, K53, I56 and I66. This fact suggests that these residues must also be related to structural functions of the LexA HTH complex rather than to the specific recognition of the DNA binding sequence. It has been suggested that, in E. coli, the third α helix of the LexA HTH complex plays the leading role in specific DNA recognition (Knegtel et al., 1995). However, other residues in the remaining αhelices or in between must also play a significant part in specific DNA recognition, since a F. succinogenes LexA protein derivative in which the sequence of the third α
helix has been replaced through directed mutagenesis with that of E. coli LexA can not bind the E. coli-like CTGTN8ACAG motif (data not shown).
Furthermore, we have also demonstrated that a functional Alpha Proteobacteria LexA binding sequence may be easily generated from the Cyanobacterial one through a single insertion event while, in turn, the Cyanobacterial LexA box derives directly from the Gram-positive one (Mazón et al., 2004). The use of DNA recognition motifs in combination with other phylogenetic evidence has been proposed earlier as a measure of divergence to refine phylogenetic analyses and as a milestone to highlight branching points in evolution (Rodionov et al., 2001; Rajewsky et al., 2002; Erill et al., 2003). Therefore, the experimental evidence of relatedness between Alpha and Cyanobacteria LexA boxes takes new relevance when combined with the fact that these two groups do also cluster together in the phylogenetic tree of LexA proteins (Fig. 5). This close relationship between Alpha Proteobacteria and Cyanobacteria is clearly at odds with the traditional positioning of the Alpha Proteobacteria class in the bacterial evolutionary tree, as prompted by RecA protein (Fig. 9; Eisen, 1995) and 16S rRNA and signature protein phylogenies (Woese et al., 1984; Gupta & Griffiths, 2002), since these three phylogenetic analyses place the Alpha Proteobacteria very close to the Beta Proteobacteria and far removed from either Cyanobacteria or Gram-positive bacteria. The most feasible explanation for this combined divergence with conventional phylogenetic data is to suppose that, after their branching from other Proteobacteria classes, Alpha Proteobacteria lost their vertically-transmitted lexA gene, but incorporated later a novel lexA copy through lateral gene transfer (LGT) from either a Cyanobacterium or a bacterial species closely related to this Phylum. This LGT addition, however, must have occurred very early in the evolutionary history of the Alpha Proteobacteria, since the same protein is present in all Alpha
Proteobacteria that have not suffered major reductions in chromosome size (e.g. Rickettsia), and GC percentage and codon usage of the extant lexA genes are in perfect agreement with the average values for each of the Alpha Proteobacteria hosting them. In this context, it should be stressed that the loss of the lexA gene does not seem to be a very unusual event in bacterial evolution, as it has already been described in several genera (such as Aquifex, Borrelia, Campylobacter, Clamydia, Helicobacter, Mycoplasma or Rickketsia). Up to now, a common characteristic of those bacteria for which the lack of a lexA gene had been described was that they had undergone a major reduction in chromosome size, suggesting that massive genome reduction was a convergent evolutionary cause for the loss of the lexA gene. However, and given that Alpha Proteobacteria species here analyzed do not present significant reductions in genetic material, our data concerning their LexA protein breaks with this traditional assumption and hints at the possible existence of losses and lateral acquisitions of the lexA gene among bacteria. Although further work is still necessary to elucidate whether similar LGT processes have taken place in other Bacterial Phyla, the reported evidence of lateral transfer of the lexA gene sheds new light on the evolutionary history of a complex regulatory network like the LexA-governed SOS response and validates the previously reported use of regulatory motifs, in combination with phylogenetic and protein signature studies, as reliable indicators of phylogenetic history.
ACKNOWLEDGEMENTS
This work was funded by Grants BMC2001-2065 from the Ministerio de Ciencia y Tecnología (MCyT) de Espa?a and 2001SGR-206 from the Departament d’Universitats, Recerca i Societat de la Informació (DURSI) de la Generalitat de
Catalunya, and by the Consejo Superior de Investigaciones Científicas (CSIC). We are deeply indebted to Dr. Roger Woodgate for his generous gifts of E. coli and B. subtilis LexA proteins. We wish to acknowledge Joan Ruiz for his excellent technical assistance and collaboration. The free access of The Institute for Genomic Research (TIGR) to the F. succinogenes preliminary sequence data is also acknowledged. Partial sequencing of F. succinogenes was accomplished with support from U.S. Department of Agriculture.
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英文标题大小写规则
英文标题大小写规则 一般实词（名词、动词、代词、形容词、副词等）首字母大写，虚词（介词、冠词、连词、感叹词）首字母小写。 标题第一个单词、最后一个单词无论词性首字母应该大写。 超过5个字母的虚词,如between、without、alongside、underneath等应该大写。 如果是重要提示性标题，或者是专有名称标题，可以全部字母都用大写，但这种用法应慎重。附： 1. 英文题名（标题）
1) 题名的结构。英文题名以短语为主要形式，尤以名词短语（noun phrase ）最常见，即题名基本上由一个或几个名词加上其前置和（或）后置定语构成。例如：Discussion About the Envy of Children an the Aged （儿童与老人之妒论略），Principles to Follow in Enrolling Talents in Higher Education Institutions （高校人才引进应遵循的原则）。短语型题名要确定好中心词，再进行前后修饰。各个词的顺序很重要，词序不当，会导致表达不准。题名一般不应是陈述句，因为题名主要起标示作用，而陈述句容易使题名具有判断式的语义；况且陈述句不够精练和醒目，重点也不易突出。少数情况（评述性、综述性和驳斥性）下可以用疑问句做题名，因为疑问句可有探讨性语
气，易引起读者兴趣。例如：Can Agricultural Mechanization be Realized Without Petroleum? （农业机械化能离开石油吗? ）。 2) 题名的字数。题名不应过长。总的原则是，题名应确切、简练、醒目，在能准确反映论文特定内容的前提下，题名词数越少越好，一般不宜超过10 个实词。专家建议不要超过15 个字，根据人的记忆特点，最好不超过12 个字，否则不易记忆，最大限度一般不超过20 个字。 3) 中英文题名的一致性。同一篇论文，其英文题名与中文题名内容上应一致，但不等于说词语要一一对应。在许多情况下，个别非实质性的词可以省略或变动。
英文金额大写规则及英语商务合同注意事项
英文金额大写规则及英语商务合同注意事项 一、英文金额大写规则 举个例子: 比如金额$116，131.37 USD的英文大写应该怎样写呢，有什么规则吗？ SAY U.S.DOLLARS ONE HUNDRED SISTEEN THOUSAND ONE HUNDRED THIRTY ONE CENTS THIRTY SEVEN ONLY 如果是人民币，则说 SAY RMB … SAY HK DOLLARS 二、英语商务合同的相关注意事项 英译商务合同貌似简单，实则不然。商务合同是一种特殊的应用文体，重在记实，用词行文的一大特点就是准确与严谨。 本文拟运用翻译教学中所积累的英译商务合同的实例，从三个方面论述如何从大处着眼、小处着手、力求准确严谨英译商务合同。(一)酌情使用公文语惯用副词 商务合同属于法律性公文，所以英译时，有些词语要用公文语词语、特别是酌情使用英语惯用的一套公文语副词，就会起到使译文结构严谨、逻辑严密、言简意赅的作用。但是从一些合同的英文译本中发现，这种公文语副词通常被普通词语所代替，从而影响到译文的质量。 实际上，这种公文语惯用副词为数并不多，而且构词简单易记。
常用的这类副词是由 here、there、where 等副词分别加上 after、by、in、of、on、to、under、upon、with 等副词，构成一体化形式的公文语副词。例如： 从此以后、今后：hereafter； 此后、以后：thereafter； 在其上：thereon、thereupon； 在其下：thereunder； 对于这个：hereto； 对于那个：whereto； 在上文：hereinabove、hereinbefore； 在下文：hereinafter、hereinbelow； 在上文中、在上一部分中：hereinbefore； 在下文中、在下一部分中：thereinafter． 现用两个实例，说明在英译合同中如何酌情使用上述副词。 例 1：本合同自买方和建造方签署之日生效。 This Contract shall come into force from the date of execution hereof by the Buyer and the Builder. 例 2：签署人特此同意在中国制造新产品，其品牌以此为合适。 The undersigned hereby agrees that the new products whereto this trade name is more appropriate are made in China. (二)慎重处理合同的关键细目 实践证明，英译合同中容易出现差错的地方，一般来说，不是大
英语单词大写规则
英语单词大写规则 It was last revised on January 2, 2021
在英语写作中，很多情况下要使用大写字母。其使用规则大致如下： (1) 所有句子的第一个单词的首字母要大写，包括引语中的句子。如： You should wear loose clothing in hot weather. He suggested, “The meeting should be put off till next.” (2) 专有名词，包括人名、地名、国名等要大写。如： Mary and Steve moved in the next door to the Johnsons. China and India are developing countries. (3) 表示种族、国籍、宗教、语言等的词语要大写。如： Chinese, Islamism, Christian, English (4) 标题中位于首尾的单词要大写；其他位置的除冠词、连词、介词外都要大写。如： Gone with the Wind. (5) 头衔放在人名前要大写；头衔表示的职位如果仅一人担任，而且头衔可以用来代替人名时，要大写。如： General George Marshall Professor Shirley Ores The Prime Minister arrives tomorrow. (6) 报纸、杂志、电影、戏剧、歌曲、书名等要大写。如： Overseas Digest A Tale of the White Snake. (7) 各种组织、俱乐部等的名字要大写。如： the National Organization for Women the Chess Club (8) 表示月份、星期、节日的名词要大写。如： Thanksgiving is celebrated on the third Thursday of November. (9) 商品的商标名要大写。如： a Sony television Kodak (10) 首字母缩写词中的所有字母都要大写。如： OPEC， NATO (11) 书信的称呼语和结尾谦辞首字母要大写。如： Dear Mr. Hill. Very sincerely yours (12) 表示亲属关系的名词在人名前时要大写。如： Aunt Mary Uncle Tom (13) 地理名词大写。如： the Midwest the South Pole (14) 表示历史阶段或历史事件的名词要大写。如： The Renaissance, The October Revolution (15) 政府机构、政党、院校名称要大写。如：
英文首字母大写规则(全)精编版
“大写”指首字母大写 “全大写”指全部字母大写。 （1）句子和标题第一个词大写。 （2）诗歌各行的首字母大写。 （3）论文大纲中各行的首字母大写。 以上三项在现代各种西文中都要大写。 （4）人名及与之连用的称呼、职称、头衔、诨号大写。如：Daniel Defoe（丹尼尔·笛福）、Oliver C. Fairbanks Jr.（小奥利弗·C. 费尔班克斯）、Uncle Tom（汤姆叔叔）、Senator Bradley （布莱德利参议员）、Lieutenant Colonel Smith(斯密斯中校)、Professor Johnson（约翰逊教授）、Stonewall Jackson（石壁杰克逊）。 原籍为爱尔兰、苏格兰等地的人有许多姓以O’、Mac、Mc为词头。如：O’Hara（奥哈拉）、MacDonald（麦克唐纳）、McKinley（麦金莱）。 英语国家法、西、葡、意、德、荷裔的人名在姓前往往带有de、della、du、der、d’、der la 、la、l’、van、van der、von、ten、ter、zur等附加成分，用大写还是小写从家族或个人习惯, 在移民的祖籍国小写的较多。不用全名，只提姓时一般要带附加成分。如：Eugen D’Albert(D’Albert)，Lee De Forest（De Forest），Walter de la Mare（de la Mare），Martin Van Braun（Van Braun），Werner von Braun（von Braun）。附加成分在句子开头大写。如：the paintings of de Kooning / De Kooning’s paintings are over there. （5）星系、恒星、行星、卫星、星座等天体名称大写。如：Milky Way（银河系）、North Star （北极星）、Saturn（土星）、Phobos（火卫一）、Ursa Major（大熊星座）。 但sun（太阳）、earth（地球）、moon（月球）通常小写，除非它们作为太阳系特定天体名称出现，或同其他大写天体名称连用，如the Moon and Mars（月球和火星）。the solar system （太阳系）也小写。 （6）海洋、海峡、港湾、岛屿、半岛、湖泊、河流、山脉、高原、平原、沙漠等自然界事物的通名作为专名的一部分时大写。如：Atlantic Ocean（大西洋）、Rocky Mountains（落基山脉）、Strait of Gibraltar（直布罗陀海峡）、Rhode Island（罗得岛）、Hudson River（哈得孙河）。 通名不作为专名的一部分时小写。如：the Caribbean islands（加勒比海诸岛）、the river Thames （泰晤士河）、the river Nile（尼罗河）。 （7）东、西、南、北、中等方位名词作为专名的一部分时大写。如：East Asia（东亚）、South Calolina（南卡罗来纳州）、the Middle East（中东）。 仅指方向时小写。如：south by east（南偏东）、west of Londun（伦敦西）。 （8）省、州、市、县、乡、区等行政区划单位名称作为专名的一部分时大写，作为普通名词时小写。如：the Province of Antario（安大略省）、Hainan Province（海南省）、Washington
英语大小写规则
英语大小写规则.txt人生重要的不是所站的位置，而是所朝的方向。不要用自己的需求去衡量别人的给予，否则永远是抱怨。一、英语文章标题大小写规则 1.题目的第一个单词要大写； 2.冠词都不需要大写； 3.字母多于三个(不含三个)的介词、连词首字母要大写； 4.名词、动词、形容词、副词、代词、感叹词首字母； 5.大写所有英语中要求大写的单词。如月份、人名、地名等等。这几条原则的优先性是递减的，也就是说，如果几条原则之间出现了矛盾的情况，应优先实用前面的原则。如：如果题目的第一个单词是冠词或不多于二个字母的介词时也应该大写。 二、英文字母大写规则 1．句子开头的第一个字母要大写。“I（我）”在句中任何位置都要大写。例如：What's her name？Mary and I are teachers． 2．地名、国名和人名等专有名词第一个字母要大写。例如：Russia（俄罗斯），Youyang（酉阳），Chengdu（成都），Jack（杰克）。 3．一些亲属关系（如mother，sister，mum，dad等）用作称呼语时第一个字母要大写。例如：Thank you，Granny．谢谢你，姥姥。 4．人名前的称呼或头衔第一个字母应大写。例如：Mr Smith，Dr Wang，Miss Mary。 5．表示语种、民族的名词或形容词第一个字母要大写。例如：Russian俄语、俄罗斯人（的），Chinese汉语、中国人（的）。 6．直接引语中，句首字母要大写。例如：“Then，”I said，“You havebeen making a mistake，and the letter is not in the apartment．”“那么，”我说，“你准弄错了。这封信并不在那栋房子里。” 7．星期、月份名称的第一个字母要大写，但季节第一个字母不大写。例如：Sunday星期天，August八月，winter冬天，spring春天。
英文大写的金额
19,800,000美金 Eighteen million eight hundred thousand U.S. Dollars 关于金额的大写： 英文中金额的大写，由三个部分组成：“SAY + 货币”+ 大写数字（amount in words）+ ONLY(相当于我们的“整”)。和汉语不同的是，数字中有零不用写出来，而是把数字读法写出来即可。 如：1 146 725.00 SAY US DOLLARS ONE MILLION ONE HUNDRED AND FORTY SIX THOUSAND SEVEN HUNDRED AND TWENTY FIVE ONLY HKD12 176 SAY HONG KONG DOLLARS TWELVE THOUSAND ONE HUNDRED AND SEVENTY SIX ONLY. 如果金额有小数，常见的有三种表达方法： 1. ...AND CENTS…(cents in words) ONLY, 如： USD 100.25 可以写成 SAY US DOLLARS ONE HUNDRED AND CENTS TWENTY FIVE ONLY 2. …AND POINT…(cents in words) ONLY. 如： JPY1 100.55 可以写成 JAPANESE YUAN ONE THOUSAND ONE HUNDRED AND POINT FIFTY FIVE ONLY 3….##/100 ONLY, 如：EUD 13 658.85可以写成 EURO DOLLARS THIRTEEN THOUSAND SIX HUNDRED AND FIFTY EIGHT 85/100 ONLY
英语大写规则
下面讲某个词语“大写”时指首字母大写，讲某个词语“全大写”时指全部字母大写。（1）句子和标题第一个词大写。 （2）诗歌各行的首字母大写。 （3）论文大纲中各行的首字母大写。 以上三项在现代各种西文中都要大写。 （4）人名及与之连用的称呼、职称、头衔、诨号大写。如：Daniel Defoe（丹尼尔·笛福）、Oliver C. Fairbanks Jr.（小奥利弗·C. 费尔班克斯）、Uncle Tom（汤姆叔叔）、Senator Bradley （布莱德利参议员）、Lieutenant Colonel Smith(斯密斯中校)、Professor Johnson（约翰逊教授）、Stonewall Jackson（石壁杰克逊）。 原籍为爱尔兰、苏格兰等地的人有许多姓以O’、Mac、Mc为词头。如：O’Hara（奥哈拉）、MacDonald（麦克唐纳）、McKinley（麦金莱）。 英语国家法、西、葡、意、德、荷裔的人名在姓前往往带有de、della、du、der、d’、der la 、la、l’、van、van der、von、ten、ter、zur等附加成分，用大写还是小写从家族或个人习惯, 在移民的祖籍国小写的较多。不用全名，只提姓时一般要带附加成分。如：Eugen D’Albert(D’Albert)，Lee De Forest（De Forest），Walter de la Mare（de la Mare），Martin Van Braun（Van Braun），Werner von Braun（von Braun）。附加成分在句子开头大写。如：the paintings of de Kooning / De Kooning’s paintings are over there. （5）星系、恒星、行星、卫星、星座等天体名称大写。如：Milky Way（银河系）、North Star （北极星）、Saturn（土星）、Phobos（火卫一）、Ursa Major（大熊星座）。 但sun（太阳）、earth（地球）、moon（月球）通常小写，除非它们作为太阳系特定天体名称出现，或同其他大写天体名称连用，如the Moon and Mars（月球和火星）。the solar system （太阳系）也小写。 （6）海洋、海峡、港湾、岛屿、半岛、湖泊、河流、山脉、高原、平原、沙漠等自然界事物的通名作为专名的一部分时大写。如：Atlantic Ocean（大西洋）、Rocky Mountains（落基山脉）、Strait of Gibraltar（直布罗陀海峡）、Rhode Island（罗得岛）、Hudson River（哈得孙河）。 通名不作为专名的一部分时小写。如：the Caribbean islands（加勒比海诸岛）、the river Thames （泰晤士河）、the river Nile（尼罗河）。 （7）东、西、南、北、中等方位名词作为专名的一部分时大写。如：East Asia（东亚）、South Calolina（南卡罗来纳州）、the Middle East（中东）。 仅指方向时小写。如：south by east（南偏东）、west of Londun（伦敦西）。 （8）省、州、市、县、乡、区等行政区划单位名称作为专名的一部分时大写，作为普通名词时小写。如：the Province of Antario（安大略省）、Hainan Province（海南省）、Washington State或the state of Washington（华盛顿州）、New England states（新英格兰各州）、New York City（纽约市）或the city of New York（纽约城）、Palm Beach County（棕榈滩县）、Evanston Township埃文斯顿乡、Seventh Congressional District（第七国会选区）或his congressional
财务中数字大写之规则
财务中数字大写之规则 汉字大写金额规则 大写金额数字到元或角为止的，在“元”或“角”字之后应写“整”或“正”字；大写金额数字有分的，分字后面不写“整”字。 大写金额数字前未印有人民币字样的，应加填“人民币”三字，“人民币”三字与金额数字之间不得留有空白。 阿拉伯金额数字中间有“０”时，汉字大写金额要写“零”字，如＄１０１．５０，汉字大写金额应写成人民币壹佰零壹圆伍角整。阿拉伯金额数字中间连续有几个“０”时，汉字大写金额中可以只写一个“零”字，如￥１，００４．５６，汉字大写金额应写成人民币壹仟零肆圆伍角陆分。阿拉伯金额数字元位是“０”，或数字中间连续有几个“０”，元位也是“０”，但角位不是“０”时，汉字大写金额可只写一个“零”字，也可不写“零”字，如＄１，３２０．５６，汉字大写金额应写成人民币壹仟叁佰贰拾圆零伍角陆分，或人民币壹仟叁佰贰拾圆伍角陆分。又如＄１，０００．５６，汉字大写金额应写成人民币壹仟圆零伍角陆分，或人民币壹仟圆伍角陆分。 支票的填写： 1、出票日期（大写）：数字必须大写，大写数字写法：零、壹、贰、叁、肆、伍、陆、柒、捌、玖、拾。 举例：2005年8月5日：贰零零伍年捌月零伍日捌月前零字可写也可
不写，伍日前零字必写。2006年2月13日：贰零零陆年零贰月壹拾叁日 (1) 壹月贰月前零字必写，叁月至玖月前零字可写可不写。拾月至拾贰月必须写成壹拾月、壹拾壹月、壹拾贰月(前面多写了“零”字也认可，如零壹拾月)。 (2) 壹日至玖日前零字必写，拾日至拾玖日必须写成壹拾日及壹拾X 日（前面多写了“零”字也认可，如零壹拾伍日，下同），贰拾日至贰拾玖日必须写成贰拾日及贰拾X日，叁拾日至叁拾壹日必须写成叁拾日及叁拾壹日。 2、收款人： (1) 现金支票收款人可写为本单位名称，此时现金支票背面“被背书人”栏内加盖本单位的财务专用章和法人章，之后收款人可凭现金支票直接到开户银行提取现金。（由于有的银行各营业点联网，所以也可到联网营业点取款，具体要看联网覆盖范围而定）。 (2) 现金支票收款人可写为收款人个人姓名，此时现金支票背面不盖任何章，收款人在现金支票背面填上身份证号码和发证机关名称，凭身份证和现金支票签字领款。 (3) 转帐支票收款人应填写为对方单位名称。转帐支票背面本单位不盖章。收款单位取得转帐支票后，在支票背面被背书栏内加盖收款单位财务专用章和法人章，填写好银行进帐单后连同该支票交给收款单位的开户银行委托银行收款。 3、付款行名称、出票人帐号：即为本单位开户银行名称及银行帐号，例如：工行高新支行九莲分理处1202027409900088888帐号小写。 4、人民币（大写）：数字大写写法：零、壹、贰、叁、肆、伍、陆、柒、捌、玖、亿、万、仟、佰、拾。 注意：“万”字不带单人旁。 举例： (1) 289,546.52 贰拾捌万玖仟伍佰肆拾陆元伍角贰分。 (2) 7,560.31 柒仟伍佰陆拾元零叁角壹分 此时“陆拾元零叁角壹分”“零”字可写可不写. (3) 532.00 伍佰叁拾贰元正 “正”写为“整”字也可以。不能写为“零角零分” (4) 425.03 肆佰贰拾伍元零叁分
英文首字母大写规则(全)
“全大写”指全部字母大写。 （1）句子和标题第一个词大写。 （2）诗歌各行的首字母大写。 （3）论文大纲中各行的首字母大写。 以上三项在现代各种西文中都要大写。 （4）人名及与之连用的称呼、职称、头衔、诨号大写。如：Daniel Defoe（丹尼尔·笛福）、Oliver C. Fairbanks Jr.（小奥利弗·C. 费尔班克斯）、Uncle Tom（汤姆叔叔）、 Senator Bradley（布莱德利参议员）、 Lieutenant Colonel Smith(斯密斯中校)、 Professor Johnson （约翰逊教授）、Stonewall Jackson（石壁杰克逊）。 原籍为爱尔兰、苏格兰等地的人有许多姓以O’、Mac、Mc为词头。如：O’Hara（奥哈拉）、MacDonald（麦克唐纳）、McKinley（麦金莱）。 英语国家法、西、葡、意、德、荷裔的人名在姓前往往带有de、della、du、der、d’、 der la 、la、l’、van、van der、 von、ten、ter、zur等附加成分，用大写还是小写从家族或个人习惯, 在移民的祖籍国小写的较多。不用全名，只提姓时一般要带附加成分。如：Eugen D’Albert(D’Albert)，Lee De Forest（ De Forest），Walter de la Mare（ de la Mare），Martin Van Braun（Van Braun），Werner von Braun（von Braun）。附加成分在句子开头大写。如：the paintings of de Kooning / De Kooning’s paintings are over there. （5）星系、恒星、行星、卫星、星座等天体名称大写。如：Milky Way（银河系）、North Star （北极星）、Saturn（土星）、Phobos（火卫一）、Ursa Major（大熊星座）。 但sun（太阳）、earth（地球）、moon（月球）通常小写，除非它们作为太阳系特定天体名称出现，或同其他大写天体名称连用，如the Moon and Mars（月球和火星）。the solar system （太阳系）也小写。
合同金额大写格式
合同金额大写格式 银行、单位和个人填写的各种票据和结算凭证是办理支付结算和现金收付的重要依据，直接关系到支付结算的准确、及时和安全。票据和结算凭证是银行、单位和个人凭以记载账务的会计凭证，是记载经济业务和明确经济责任的一种书面证明。因此，填写票据和结算凭证必须做到标准化、规范化、要素齐全、数字正确、字迹清晰、不错漏、不潦草、防止涂改。 中文大写金额数字应用正楷或行书填写，如壹、贰、叁、肆、伍、陆、柒、捌、玖、拾、佰、仟、万、亿、元、角、分、零、整(正)等字样，不得用一、二(两)、三、四、五、 六、七、八、九、十、毛、另(或0)填写，不得自造简化字。如果金额数字书写中使用繁体字，也应受理。 人民币大写的正确写法还应注意以下几项： 一、中文大写金额数字到“元”为止的，在“元”之后、应写“整”(或“正”)字；在“角”之后，可以不写“整”(或“正”)字；大写金额数字有“分”的，“分”后面不写“整”(或“正”)字。
二、在票据和结算凭证大写金额栏内不得预印固定的“仟、佰、拾、万、仟、佰、拾、元、角、分”字样。 三、阿拉伯数字小写金额数字中有“0”时，中文大写应按照汉语语言规律、金额数字构成和防止涂改的要求进行书写。举例如下： 1、阿拉伯数字中间有“0”时，中文大写要写“零”字，如￥1409.50应写成人民币壹仟肆佰零玖元伍角； 2、阿拉伯数字中间连续有几个“0”时、中文大写金额中间可以只写一个“零”字，如￥6007.14应写成人民币陆仟零柒元壹角肆分。 3、阿拉伯金额数字万位和元位是“0”，或者数字中间连续有几个“0”，万位、元位也是“0”但千位、角位不是“0”时，中文大写金额中可以只写一个零字，也可以不写“零”字，如￥1680.32应写成人民币壹仟陆佰捌拾元零叁角贰分，或者写成人民币壹仟陆佰捌拾元叁角贰分。又如￥107000.53应写成人民币壹拾万柒仟元零伍角叁分，或者写成人民币壹拾万零柒仟元伍角叁分。 4、阿拉伯金额数字角位是“0”而分位不是“0”时，中文大写金额“元”后面应写“零”字，如￥16409.02应写成人民币壹万陆仟
中英合同金额数字的表达
合同金额数字的表达 Expression of Amount in Contract 英文中金额的大写，由三个部分组成：“SAY + 货币”+ 大写数字（amount in words）+ ONLY(相当于我们的“整”)。和汉语不同的是，数字中有零不用写出来，而是把数字读法写出来即可。 如： 1 146 725.00 美元 SAY US DOLLARS ONE MILLION ONE HUNDRED AND FORTY SIX THOUSAND SEVEN HUNDRED AND TWENTY FIVE ONLY HKD12 176 SAY HONG KONG DOLLARS TWELVE THOUSAND ONE HUNDRED AND SEVENTY SIX ONLY. 18,800,000美金Eighteen million eight hundred thousand U.S. Dollars 如果金额有小数，常见的有三种表达方法： 1. ...AND CENTS…(cents in words) ONLY, 如：USD 100.25 可以写成 SAY US DOLLARS ONE HUNDRED AND CENTS TWENTY FIVE ONLY 2. …AND POINT…(cents in words) ONLY. 如：JPY1 100.55 可以写成 JAPANESE YUAN ONE THOUSAND ONE HUNDRED AND POINT FIFTY FIVE ONLY 3 EUD 13 658.85可以写成 EURO DOLLARS THIRTEEN THOUSAND SIX HUNDRED AND FIFTY EIGHT 85/100 ONLY 英文金额表达式中，小数点前每三位必须有一个逗号，靠近小数点的第一个逗号是Thousand（千), 以此类推，第二个逗号是Million （百万），第三个逗号是Billion（十亿）；而且英文中小数点后面的数字都是按单个数字来读，不连在一起读。 SAY US DOLLARS (...........) ONLY. 例如：USD1,234,567.89 SAY US DOLLARS ONE MILLION AND TWO HUNDRED THIRTY-FOUR THOUSAND AND FIVE HUNDRED SIXTY-SEVEN a million two hundred thirty-four thousand and five hundred and sixty-seven point eight nine dollars million:百万thousand:千point:点“a m illion”是“100万”，由于英文是没有“十万、万”这两个单位的，所以说“、十万、万”的时候用英文是用“千”作为单位，也就是“100个千、10个千”，所以上面就是“two h undred t hirty-four t housand （234个千）”。 Amount:$1,234,567.89(Say U .S .Dollars One Million Two Hundred Thirty-four Thousand And Five Hundred And Sixty-seven Point Eight only) USD240,000.00 SAY US DOLLARS TWO HUNDRED AND FORTY THOUSAND ONLY. USD2755.00 SAY US DOLLARS TWO THOUSAND SEVEN HUNDRED AND FIFTY-FIVE ONLY EUR4768.36 SAY EUROPEAN DOLLARS FOUR THOUSAND SEVEN HUNDRED
阿拉伯数字转换成英文大写
启动Microsoft Excel。 按Alt+F11 启动Visual Basic 编辑器。 在“插入”菜单上，单击“模块”。 在模块表中键入下面的代码。 Option Explicit 'Main Function Function SpellNumber(ByVal MyNumber) Dim Dollars, Cents, Temp Dim DecimalPlace, Count ReDim Place(9) As String Place(2) = " Thousand " Place(3) = " Million " Place(4) = " Billion " Place(5) = " Trillion " ' String representation of amount. MyNumber = Trim(Str(MyNumber)) ' Position of decimal place 0 if none. DecimalPlace = InStr(MyNumber, ".") ' Convert cents and set MyNumber to dollar amount. If DecimalPlace > 0 Then Cents = GetTens(Left(Mid(MyNumber, DecimalPlace + 1) & _ "00", 2)) MyNumber = Trim(Left(MyNumber, DecimalPlace - 1)) End If Count = 1
Do While MyNumber <> "" Temp = GetHundreds(Right(MyNumber, 3)) If Temp <> "" Then Dollars = Temp & Place(Count) & Dollars If Len(MyNumber) > 3 Then MyNumber = Left(MyNumber, Len(MyNumber) - 3) Else MyNumber = "" End If Count = Count + 1 Loop Select Case Dollars Case "" Dollars = "No Dollars" Case "One" Dollars = "One Dollar" Case Else Dollars = Dollars & " Dollars" End Select Select Case Cents Case "" Cents = " and No Cents" Case "One" Cents = " and One Cent" Case Else Cents = " and " & Cents & " Cents"
英文标题大小写规则
英文标题大小写规则 一般实词(名词、动词、代词、形容词、副词等)首字母大写,虚词(介词、冠词、连词、感叹词)首字母小写。 标题第一个单词、最后一个单词无论词性首字母应该大写。 超过5个字母得虚词,如between、without、alongside、underneath等应该大写。 如果就是重要提示性标题,或者就是专有名称标题,可以全部字母都用大写,但这种用法应慎重。附: 1、英文题名(标题) 1) 题名得结构。英文题名以短语为主要形式,尤以名词短语( noun phrase )最常见,即题名基本上由一个或几个名词加上其前置与(或)后置定语构成。例如: Discussion About the Envy of Children an the Aged (儿童与老人之妒论略), Principles to Follow in Enrolling Talents in Higher Education Institutions (高校人才引进应遵循得原则)。短语型题名要确定好中心词,再进行前后修饰。各个词得顺序很重要,词序不当,会导致表达不准。题名一般不应就是陈述句,因为题名主要起标示作用,而陈述句容易使题名具有判断式得语义;况且陈述句不够精练与醒目,重点也不易突出。少数情况(评述性、综述性与驳斥性)下可以用疑问句做题名,因为疑问句可有探讨性语气,易引起读者兴趣。例如: Can Agricultural Mechanization be Realized Without Petroleum? (农业机械化能离开石油吗? )。 2) 题名得字数。题名不应过长。总得原则就是,题名应确切、简练、醒目,在能准确反映论文特定内容得前提下,题名词数越少越好,一般不宜超过10 个实词。专家建议不要超过15 个字,根据人得记忆特点,最好不超过12 个字,否则不易记忆,最大限度一般不超过20 个字。 3) 中英文题名得一致性。同一篇论文,其英文题名与中文题名内容上应一致,但不等于说词语要一一对应。在许多情况下,个别非实质性得词可以省略或变动。 4) 题名中得大小写。题名字母得大小写有以下三种格式。A、全部字母大写。例如: DISCUSSION ABOUT THE ENVY OF CHIDREN AND THE AGED 。B、每个词得首字母大写,但三个或四个字母以下得冠词、连词、介词全部小写。例如: From “Gobacktohistory” to Nonhistory — A Criticism of New Historicism 。C、题名第一个词得首字母大写,其余字母均小写。例如: Topographic inversion of interval velocities .一般采用B、格式。 2、作者与作者单位得英译 1) 作者。中国人名按汉语拼音拼写。中国作者姓名得汉语拼音采用如下写法:姓前名后,中间为空格, 例如: Li Ping (李平) Li Xiaoping (李小平)。 2) 单位。单位名称要写全(由小到大)。例如: No、152, Xingan West Road, Guangzhou, Guangdong 。地名得拼写方法就是:第一个字得头字母大写,后面得字紧跟在后面小写,例如: Beijing, Nanhai 。 3、英文摘要
英文标题大写规则
Capitalization in Titles 英文标题大写规则 NIVA follows the general rules for capitalizing words in document titles set out in The Chi cago Manual of Style (with one minor exception—see the note in rule 3): 在《芝加哥风格指南》中，尼瓦遵循了书写文献标题时应当一贯遵守的字母大写规则。（一些特例将在第三条规则中被加以讨论） 1.Always capitalize the first and the last word. 标题首末的两个单词必须大写。 2. 2. Capitalize all nouns, pronouns, adjectives, verbs, adverbs, and subordinate conjunctions ("as", "because", "although"). 所有的名词、代词、形容词、动词、副词和从属连词（as,because,althrough）必须大写。 3.Lowercase all articles, coordinate conjunctions ("and", "or", "nor"), and prepositions regardless of length, when they are other than the first or last word. (Note: NIVA prefers to capitalize prepositions of five characters or more ("after", "among", "between").) 所有的文献名、并列名词、介词（不论长短）在不违反第一条规则的情况下都必须小写。 4.Lowercase the "to" in an infinitive. 不定时中的to要小写。（补充：尼瓦更倾向于对由超过4个字母组成的介词采用大写形式，例 如after,aong,between） Most writers are familiar with these general rules. But some have difficulty identifying the various parts of speech, while others have internalized incorrect "rules" taught in elementary school. These individuals are therefore prone to making mistakes when capitalizing or lowercasing words in titles. The most common mistakes are presented below. 大多数书写者对于这些规则是熟悉的。但有的人存在着识别句子成分的困难；有的人深受小学时错误的引导。因而这些人在标题单词大写小写的问题上容易出错。以下是一些最常见的错误。 Two-Letter Words Some writers lowercase all two-letter words, probably by extrapolation from the short prepositions "of", "to", "up", and so on, and the word "to" in infinitives. But if a two-letter word is acting as a noun, pronoun, adjective, or adverb, it must be capitalized. For example:
英文金额大写规则及英语商务合同注意事项
英文金额大写规则 举个例子: 比如金额116131.37的英文大写应该怎样写呢，有什么规则吗？ SAY DOLLARS ONE HRNDRED SIXTEEN THOUSNAD ONE HUNDRED THIRTY ONE AND THIRTY SEVEN CENTS ONLY 如果是美元则SAY US DOLLARS。。 SAY RMB 。。。。 SAY HK DOLLARS 下面是英语商务合同的相关注意事项 英译商务合同貌似简单，实则不然。商务合同是一种特殊的应用文体，重在记实，用词行文的一大特点就是准确与严谨。 本文拟运用翻译教学中所积累的英译商务合同的实例，从三个方面论述如何从大处着眼、小处着手、力求准确严谨英译商务合同。 一、酌情使用公文语惯用副词 商务合同属于法律性公文，所以英译时，有些词语要用公文语词语、特别是酌情使用英语惯用的一套公文语副词，就会起到使译文结构严谨、逻辑严密、言简意赅的作用。但是从一些合同的英文译本中发现，这种公文语副同常被普通词语所代替，从而影响到译文的质量。 实际上，这种公文语惯用副同为数并不多，而已构词简单易记。常用的这类副词是由here、there、where 等副词分别加上after、by、in、of、on、to、under、upon、
with 等副词，构成一体化形式的公文语副词。例如： 从此以后、今后：hereafter； 此后、以后：thereafter； 在其上：thereonthereupon； 在其下：thereunder； 对于这个：hereto； 对于那个：whereto； 在上文：hereinabovehereinbefore； 在下文：hereinafterhereinbelow； 在上文中、在上一部分中：thereinbefore； 在下文中、在下一部分中：thereinafter． 现用两个实例，说明在英译合同中如何酌情使用上述副词。 例1：本合同自买方和建造方签署之日生效。 This Contract shall come into force from the date of execution hereof by the Buyer and the Builder. 例2：下述签署人同意在中国制造新产品，其品牌以此为合适。 The undersigned hereby agrees that the new products whereto this trade name is more appropriate are made in China. 二、慎重处理合同的关键细目 实践证明，英译合同中容易出现差错的地方，一般来说，不是大的陈述性条款。而恰恰是一些关键的细目．比如：金钱、时间、数量等。为了避免出差错，在英译合同时，常常使用一些有限定作用的结构来界定细目所指定的确切范围。