Evolution of a Protein-Folding Machine Genomic and Evolutionary Analyses
Evolution of a Protein-Folding Machine:Genomic and Evolutionary Analyses Reveal Three Lineages of the Archaeal hsp70(dnaK )Gene
Alberto J.L.Macario,1,2Luciano Brocchieri,3Avinash R.Shenoy,4Everly Conway de Macario 1
1
Division of Molecular Medicine,Wadsworth Center,Room B-749,New YorkState Department of H ealth,Empire State Plaza P.O.Box 509,Albany,NY 12201-0509,USA 2
Department of Biomedical Sciences,School of Public H ealth,State University of New York(SUNY)at Albany,Albany,NY 12201-0509,USA 3
Department of Mathematics,Stanford University,Stanford,CA 94305-2125,USA 4
Department of Molecular Reproduction,Development and Genetics,Indian Institute of Science,Bangalore 560012,India Received:30August 2005/Accepted:14March 2006[Reviewing Editor :Dr.Stephen Freeland]
Abstract.The stress chaperone protein Hsp70(DnaK)(abbreviated DnaK)and its co-chaperones Hsp40(DnaJ)(or DnaJ)and GrpE are universal in bacteria and eukaryotes but occur only in some ar-chaea clustered in the order 5￠-grpE-dnaK-dnaJ-3￠in a locus termed Locus I.Three structural varieties of Locus I,termed Types I,II,and III,were identi?ed,respectively,in Methanosarcinales,in Thermoplas-matales and Methanothermobacter thermoautotrophi-cus ,and in Halobacteriales.These Locus I types corresponded to three groups identi?ed by phyloge-netic trees of archaeal DnaK proteins including the same archaeal subdivisions.These archaeal DnaK groups were not signi?cantly interrelated,clustering instead with DnaKs from three bacterial lineages,Methanosarcinales with Firmicutes,Thermoplasma-tales and M.thermoautotrophicus with Thermotoga ,and Halobacteriales with Actinobacteria,suggesting that the three archaeal types of Locus I were acquired by independent events of lateral gene transfer.These associations,however,lacked strong bootstrap sup-port and were sensitive to dataset choice and tree-reconstruction method.Structural features of dnaK loci in bacteria revealed that Methanosarcinales and Firmicutes shared a similar structure,also common to most other bacterial groups.Structural di?erences
were observed instead in Thermotoga compared to Thermoplasmatales and M.thermoautotrophicus ,and in Actinobacteria compared to Halobacteriales.It was also found that the association between the DnaK sequences from Halobacteriales and Actino-bacteria likely re?ects common biases in their amino acid compositions.Although the loci structural features and the DnaK trees suggested the possibility of lateral gene transfer between Firmicutes and Methanosarcinales,the similarity between the ar-chaeal and the ancestral bacterial loci favors the more parsimonious hypothesis that all archaeal sequences originated from a unique prokaryotic ancestor.Key words:hsp70(dnaK)locus —Archaeal molec-ular chaperones —Genome analysis —Protein folding evolution —Stress genes
Introduction
Survival depends to a signi?cant extent on the ability to adapt to new situations such as sudden changes in the environment.These changes,or stressors,may involve temperature,pH,pressure,salinity,chemi-cals,and other conditions,and cause cell stress.If the changes exceed a certain degree of intensity and/or
Correspondence to:Everly Conway de Macario;email:everlym@
https://www.wendangku.net/doc/eb5443111.html,
J Mol Evol (2006)63:74–86
DOI:10.1007/s00239-005-6207-1
duration,the cell stress may reach a dangerous level and may cause cell damage or even death(Geor-gopoulos et al.1990;Linquist1992;Nollen and Morimoto2002).Cells have mechanisms to deal with stress and its consequences(e.g.,protein denatur-ation);these mechanisms include the chaperoning systems(Missiakas et al.1996;Linquist1992;Bukau et al.2000;Macario and Conway de Macario2001; Young et al.2004),which exist in all organisms and in all cells,tissues,and organs of multicellular life forms(Gupta1995;1998;Gupta and Golding1996; Bustard and Gupta1997;Gupta et al.1997;Karlin and Brocchieri1998;Willison1999;Brocchieri and Karlin2000;Karlin and Brocchieri2000;Laksan-alamai et al.2004;Kultz2005).The universality of the chaperoning systems testi?es to their importance and,also,shows that assisted protein folding,as well as refolding of denatured or misfolded polypeptides, is essential for life.We may then infer that healthy chaperoning systems must be maintained at all times, sickchaperoning systems must be restored to nor-mality as rapidly as possible lest the cell succumb to stress,and manipulation of these systems(e.g.,by means of genetic engineering)o?ers a potentially powerful means for invigorating cells so as to prevent the ravages of stress and to enhance recovery after stress.In view of the latter promising possibilities,we focused on chaperoning systems.We have applied a multidisciplinary approach in various areas;one of these areas includes the elucidation of the distribution of four de?ned chaperoning systems among organ-isms of the phylogenetic domain Archaea,and a comparison between this and the other two domains, Bacteria and Eucarya(eukaryotes).The chaperoning systems investigated were the molecular chaperone machine,chaperonins of group I,chaperonins of group II,prefoldins,and associated co-chaperones or co-factors(Macario and Conway de Macario2001; Macario et al.2004).The molecular chaperone ma-chine is essentially composed of three proteins, Hsp70(DnaK),Hsp40(DnaJ)(abbreviated DnaK and DnaJ,respectively),and GrpE in prokaryotes (Georgopoulos et al.1990;Bukau et al.2000;Har-rison2003),but in eukaryotes GrpE is replaced by other molecules(Alberti et al.2003).
A pivotal?nding was the demonstration that some archaea possess the molecular chaperone machine, but others do not(Macario and Conway de Macario 1999).This was surprising,because the machine is present in all bacterial and eukaryotic organisms, with no exception reported to date.
Absence of the chaperone machine in some ar-chaeal organisms raised fundamental questions: What are the origin and evolution of the archaeal hsp70(dnaK)(in short dnaK)and its two companion genes,hsp40(dnaJ)(abbreviated dnaJ)and grpE? And what can the structural organization of these genes reveal about their mode of transcription and regulation?We have addressed these questions in the workreported here.
The dnaK locus?rst described for an archaeon was organized5￠-hsp16-grpE-dnaK-dnaJ-trkA-3￠(Conway de Macario et al.1994;Macario et al.1999),sug-gesting that this gene arrangement might be the norm in archaea(Macario and Conway de Macario1999). Also,it was suggested that archaeal DnaK sequences are not obviously di?erentiated from bacterial counterparts(Karlin and Brocchieri1998)and that the archaeal dnaK genes originated by lateral gene transfer from bacteria(Gribaldo et al.1999).
To elucidate the origin and evolution of the genes encoding the molecular chaperone machine in the Archaea,we have carried out genomic and phyloge-netic analyses.We examined the genes encoding DnaK and the other two components of the chaper-one machine,DnaJ and GrpE,to determine the characteristics of their loci and to search for clues about their transcriptional modes.In parallel,we performed extensive phylogenetic analyses of DnaK proteins.
Materials and Methods
Genome Analyses
The loci and genes studied from the three phylogenetic domains were found in the databases and at the genome-sequence Web sites (see Web Site References);we used accession numbers when available,annotation lists,BLAST searches,and visual-manual searches of genome-sequence displays.The start and end of each coding region,and the lengths of the coding and intergenic regions, were recorded,and the data were organized in sequence from left to right,in the5￠-to-3￠direction.More information about the sources of sequences is provided in the?gure legends.
Web Sites
Web sites most frequently used are listed under Web Site Refer-ences(see Supplemental Material online).
Sequence Comparisons and Analyses
Comparative analyses of sequences were performed using the GCG (Genetics Computer Group,University of Wisconsin)and Accel-rys,Inc.(San Diego,CA),software.Analyses of amino acid con-tent were performed considering the frequency of amino acid types in conserved regions of DnaK sequences obtained as described below.Amino acids were classi?ed based on the G+C content of their codons(G+C rich—Ala,Arg,Gly,Pro;A+T rich—Ile,Lys, Asn,Tyr,Phe;others—Asp,Glu,His,Leu,Met,Gln,Ser,Thr, Val,Trp),or based on the amino acid preferences in thermophilic organisms(Glu,Gly,Arg,Val,Tyr)or in mesophilic organisms (Cys,His,Asn,Gln,Ser,Thr),and constructing two di?erent three-letter alphabets.Pairwise distance measures between groups of sequences were computed as the sum of the absolute di?erence in amino acid type frequencies and were used to cluster the sequences with the average-linkage method(Sokal and Rolf1981).
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Phylogenetic Analyses
We analyzed the phylogenetic relations of15conserved archaeal Hsp70(DnaK)sequences with eukaryotic and bacterial DnaK se-quences.From an initial set that included327archaeal,bacterial, and eukaryotic Hsp70(DnaK)sequences that we were able to col-lect from public databases,we selected198sequences(available in Supplemental Material,Table S1,online),applying the following criteria.Among genes from prokaryotic species,whenever multiple copies of dnaK genes were present in one genome we selected among them the most conserved paralogue,i.e.,the gene copy that was less diverged from their common ancestor.We excluded all eukaryotic organellar sequences and we eliminated all sequences with anomalously high or low divergence rates,as determined from preliminary trees.We also excluded bacterial and eukaryotic se-quences that branched in unexpected positions so as to avoid inclusion of possibly misannotated sequences.The?nal set in-cluded15archaeal,81bacterial,and102eukaryotic sequences. Selection of a reduced set allowed us(i)to obtain a more reliable alignment of the sequences;(ii)to minimize the e?ect of long-branch and short-branch attraction associated with fast-diverging sequences;(iii)to minimize the problem of heterogeneous evolu-tionary rates in di?erent lineages when inferring position-speci?c evolutionary rates;and(iv)to increase the number of aligned positions used for phylogenetic tree reconstructions from252 positions in the original set to465positions in the?nal set of198 sequences.Besides a preliminary tree of all327sequences(shown in Supplemental Material,Fig.S1,online),phylogenetic trees were constructed for(i)96prokaryotic plus102eukaryotic sequences; (ii)96prokaryotic sequences;(iii)a set of33sequences including all 15archaeal sequences,16bacterial sequences representative of16 distinct bacterial phyla,and2eukaryotic sequences representative of cytoplasmic and endoplasmatic reticulum(ER)sequences for computationally intensive maximum-likelihood(ML)analyses;(iv) 21‘‘consensus sequences’’obtained with the sequence alignment program ITERALIGN(Brocchieri and Karlin1998),representing 3archaeal,16bacterial and2eukaryotic groups(see Results);and (v)the19‘‘consensus sequences’’representative of prokaryotic groups only.Alignments were obtained with the multiple sequence alignment program Clustal-W(Thompson et al.1994).Phyloge-netic trees were calculated using the neighbor-joining(NJ)algo-rithm(Saitou and Nei1994),the maximum likelihood heuristic algorithm quartet puzzling(QP)(Strimmer and Haeseler1996)as implemented in the computer program TREE-PUZZLE(Schmidt et al.2002),and the maximum likelihood(ML)algorithm(Kishino et al.1990)as implemented in the PHYLIP package(Felsenstein 1989).Pairwise-distance matrices were estimated assuming gamma-distributed mutational rates(Ota and Nei1994),with the param-eter a calculated with the ML procedure implemented in the phylogenetic program TREE-PUZZLE(Schmidt et al.2002). Bootstrap values were based on100samples for ML trees and for the preliminary NJ tree with327sequences and on1000samples for all other trees.
Results
dnaK Loci in Archaea
We carried out a search of electronic and printed literature,and of sequenced genomes,to identify all the sequenced dnaK genes present in archaeal species. The identi?ed genes were mapped along with the ?anking genes,whenever su?cient and reliable se-quence information was available.Examination of the structure and organization of the archaeal dnaK loci revealed that these loci could be classi?ed into three types,termed Types I,II and III.
Locus I Types I and II were both characterized by the typical triad arrangement5￠-grpE-dnaK-dnaJ-3￠. However,in Type I,the intergenic region between grpE and dnaK was considerably longer(78to431bp) than that in Type II()23to14bp)(Figs.1A and B). The structure of the triad in the loci from Methano-sarcina species was quite conserved(Fig.1A),as was the structure of the triad in the Thermoplasmatales (Fig.1B).The dnaK locus of Picrophilus torridus (Futterer et al.2004)is organized as in other Ther-moplasmatales(Thermoplasma and Ferroplasma),and the intergenic region between the?rst two genes is)23 bp long(i.e.,grpE and dnaK overlap over23bp).This overlap corresponds to an extension of nine amino acids of the C-terminal tail of the P.torridus DnaK protein compared to the protein in Thermoplasma species.The intergenic region between dnaK and dnaJ in P.torridus is8bp long(Fig.1B).Taken together, these data show that the intergenic regions in these organisms are very short,or nonexistent,as is that between grpE and dnaK in P.torridus.In Locus I from Methanothermobacter thermoautothrophicus the dis-tance between dnaK and dnaJ(150bp)resembled the intergenic distance observed in the Type I Locus I of Methanosarcinales(66–129bp)(Fig.1A)or the in-tergenic distance observed in the Type III Locus I of Halobacteriales(75–141bp)(Fig.1C).However,the short intergenic region between grpE and dnaK(14 bp)was typical of Locus I Type II()23to6bp) (Fig.1B).Phylogenetic analyses of DnaK proteins (see below)supported the classi?cation of M.ther-moautotrophicus Locus I as Type II.
A diversi?ed pattern was observed for Locus I Type III.In Halobacterium sp.NRC-1and Haloferax mediterranei(Fig.1C),grpE and dnaK were sepa-rated by891and1135bp,respectively,with one predicted gene between them.In these genomes,dnaK and dnaJ were encoded next to each other with sep-arations of75–141bp,similarly to Locus I Type I.In Haloarcula marismortui,grpE and dnaK were sepa-rated by165bp,without any gene between them,as in the loci of Types I and II,but the third member of the triad,dnaJ,was encoded over3000bp down-stream of dnaK,separated from it by four predicted genes.These results showed more di?erences between loci among Halobacteriales than among Methanos-arcinales or Thermoplasmatales.However,phyloge-netic analysis of DnaK proteins con?rmed the close relation of these loci,which should be considered as one diversi?ed group(see below).
dnaK Locus I Types and DnaK Phylogeny
The evolutionary relations of archaeal Locus I among Archaea and its relation with bacterial loci can in
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principle be studied by the relations among its gene or protein sequences.Among these,grpE and dnaJ have evolved at a fast rate and cannot be reliably used for phylogenetic analyses when comparing archaeal and bacterial genes/proteins.Genes for DnaK are instead quite conserved even between Archaea and Bacteria and their evolutionary relations can be used to rep-resent the evolutionary relations of the corresponding loci.
Phylogenetic trees of DnaK protein sequences were constructed with di?erent procedures and col-lections of sequences (see Materials and Methods).Results of these analyses relevant to the evolution of archaeal sequences are summarized in Table https://www.wendangku.net/doc/eb5443111.html,plete NJ phylogenetic trees for a set of 198eukaryotic and prokaryotic DnaK sequences and for a set of 96prokaryotic sequences are shown in Figs.2and 3,https://www.wendangku.net/doc/eb5443111.html,plete trees obtained with other collections of sequences (see Materials and Methods)and/or with the ML method are shown in
the Supplemental Material,Figs.S2–S6,online.A largely unresolved tree was produced by the QP procedure.Clusters obtained by this procedure with the highest reliability are listed in the condensed TREE-PUZZLE output ?le (see Supplemental Material,Output of TREE-PUZZLE,online).All trees supported with high reliability the clustering of archaeal DnaK sequences into three separate groups:Group 1included all sequences from Methanosarci-nales (which have Locus I of Type I);Group 2included all sequences from Thermoplasmatales (from the genera Thermoplasma ,Ferroplasma ,and Picrophilus ,which have Locus I of Type II)and,in addition,the sequence from M.thermoautotrophicus ,supporting the classi?cation of its Locus I as Type II;Group 3included all sequences from
Halobacteriales,
Fig.1.A dnaK Locus I Type I.Schematic of the extended dnaK loci in Methanosarcina acetivorans C2A (M.a.),Methanosarcina mazeii S-6(M.m.6),Methanosarcina mazeii Goe1(M.m.1),Methanosarcina barkeri (M.ba.),Methanosarcina thermophila TM1(M.th.),and Methanococcoides burtonii (M.bu.).The loci are shown from 5￠to 3￠,from left to right.Each box represents a gene and the ?gures underneath each row of genes represent the length,in base pairs (bp),of the respective intergenic regions.The loci were aligned one underneath another by placing the dnaK genes in a single column.The M.acetivorans C2A locus was used as reference and is shown in the top row,where the genes ?names are displayed above their respective boxes.Boxes with a name or a numerical designation inside represent genes that are di?erent from the gene at the top of the respective column and that have been annotated and assigned a putative function.NN indicates that the gene has no known function or speci?c name.Gene 3is dnaK ,i.e.,the central gene that de?nes the locus.This locus includes the stress-gene triad composed of genes 2(three-celled box),3(four-celled box),and 4(two-celled box),namely,5￠-grpE-dnaK-dnaJ -3￠.The other genes on both sides of the triad,when known,are displayed,to show that they are not as conserved as the stress-gene triad.Only 159bp has been sequenced at the 3￠end of grpE in M.thermophila TM1.B dnaK Locus I Type II.The loci shown are from Thermoplasma acidophilum (T.a.),Thermoplasma volcanium (T.v.),Ferroplasma acidarmanus (F.a.),Picrophilus torridus (P.t.),and Methanoth-ermobacter thermoautotrophicus (Methanobacterium thermoauto-trophicum)Delta H (M.t.).C dnaK Locus I Type III.The loci shown are from Halobacterium sp.NRC-1(H.s.)and from Haloferax mediterranei (H.m.).Org.,organism;perm.,permease;RNAP,RNA polymerase;k-ase,kinase;RNaseP,RNaseP RNA.The sequence sources were the genome Web sites (see Web Site References in Supplemental Material,Table S3,online)and Con-way de Macario et al.1994,Gupta and Singh 1993,Macario et al.1991,1993,1995,Ho?man-Bang et al.1999,Deppenmeir et al.2002,Galagan et al.2002,Smith et al.1997(M.thermoautotrohi-cum Delta H),Kawashima et al.2000(T.volcanium ),Ruepp et al.2000(T.acidophilum ),Futterer et al.2004(P.torridus ),Ng et al.2000(Halobacterium sp.NRC-1),and A.S.Kazi and C.K.K.Nair,Bombay,India (accession number AF069527;gi,10798841;H.mediterranei ).For H.mediterranei ,no sequence is available up-stream of grpE .Not shown is the locus from Haloarcula maris-mortui (whose genome has recently been sequenced [Baliga et al.2004;https://www.wendangku.net/doc/eb5443111.html,;for sequence,data,annota-tions,and analyses,https://www.wendangku.net/doc/eb5443111.html,/interpro/READ-ME1.html;InterProScan,http://www.genome.ad.jp/kegg-bin/srch_orth_html;KEGG database,https://www.wendangku.net/doc/eb5443111.html,/COG]),in which the dnaJ gene is not in the dnaK locus but is separated from the latter by 3266bp,with other genes between them.grpE and dnaK are consecutive genes (no other gene between them),sepa-rated by 165bp.
b
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T a b l e 1.P e r c e n t a g e b o o t s t r a p s u p p o r t o f m i x e d b a c t e r i a l a n d a r c h a e a l c l u s t e r s
N J N J N J c o n s N J c o n s Q P M L M L c o n s M L c o n s 102e u k +96p r o k 96p r o k 2e u k +19p r o k 19p r o k 96p r o k 2e u k +31p r o k 2e u k +19p r o k 19p r o k
M e t h a n o s a r c i n a l e s a
100100n .a .n .a .79100n .a .n .a .H a l o b a c t e r i a l e s 100100n .a .n .a .9499n .a .n .a .T h e r m o p l a s m a t a l e s 100100n .a .n .a 7794n .a .n .a .T h e r m o p l a s m a t a l e s +M e t h a n o t h e r m o b a c t e r 98100n .a .n .a .6898n .a .n .a .M e t h a n o s a r c i n a l e s +F i r m i c u t e s 72b
6642b
2549b
46(26b ,c )(25b ,c )H a l o b a c t e r i a l e s +A c t i n o b a c t e r i a (18)403541—d
23(8)(6)T h e r m o p l a s m a t a l e s +M e t h a n o t h e r m o b a c t e r +A q u i f e x +T h e r m o t o g a (4)12(5)(4)—(11)2931T h e r m o p l a s m a t a l e s +M .t h e r m o a u t o t r o p h i c u s +T h e r m o t o g a 33(23)(21)(22)—(33)c
5755H a l o b a c t e r i a l e s +T h e r m o p l a s m a t a l e s +M e t h a n o t h e r m o b a c t e r +T h e r m o t o g a 26(19)(27)(26)—(12)(5)(14)T h e r m o p l a s m a t a l e s +M e t h a n o t h e r m o b a c t e r +D e i n o c o c c i +C y a n o b a c t e r i a +C h l o r o ?e x a l e s +A q u i f e x +T h e r m o t o g a (1)
(14)(2)11—(2)(11)—
T h e r m o p l a s m a t a l e s +M e t h a n o t h e r m o b a c t e r +E u k a r y a
(1)n .a .16n .a .n .a .(5)—
n .a .
N o t e .N J ,n e i g h b o r -j o i n i n g m e t h o d ;Q P ,q u a r t e t p u z z l i n g m e t h o d ;M L ,m a x i m u m l i k e l i h o o d m e t h o d ;c o n s ,c o n s e n s u s s e q u e n c e s ;e u k ,e u k a r y o t i c s e q u e n c e s ;p r o k ,p r o k a r y o t i c s e q u e n c e s ;n .a .,n o t a p p l i c a b l e .V a l u e s i n p a r e n t h e s e s i n d i c a t e b o o t s t r a p s u p p o r t f o r c l u s t e r s n o t a p p e a r i n g i n t h e t r e e s o b t a i n e d f r o m t h e o r i g i n a l d a t a .F o r Q P t h e i n d i c a t e d v a l u e s r e p r e s e n t p e r c e n t a g e ‘‘r e l i a b i l i t y v a l u e s ’’o b t a i n e d f r o m 50,000r a n d o m i z a t i o n s o f t h e t a x o n i n p u t o r d e r i n t h e p u z z l i n g s t e p o f t h e p r o c e d u r e .a A r c h a e a l g r o u p s a r e i n b o l d f a c e .b I n c l u d i n g a l s o a s e q u e n c e f r o m F u s o b a c t e r i u m .c G r o u p o b s e r v e d i n t h e M L c o n s e n s u s t r e e (a s o b t a i n e d f r o m t h e p r o g r a m C O N S E N S E o f t h e P h y l i p p a c k a g e w i t h t h e m a j o r i t y -r u l e -e x t e n d e d o p t i o n ).d A d a s h i n d i c a t e s t h a t t h e c l u s t e r w a s n o t o b s e r v e d a m o n g t h e b o o t s t r a p p e d r e p l i c a s o r ,i n t h e c a s e o f t h e Q P p r o c e d u r e ,t h e c l u s t e r w a s o b s e r v e d t o o i n f r e q u e n t l y t o b e r e p o r t e d (r e l i a b i l i t y v a l u e s l e s s t h a n 30%).
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supporting the classi?cation of their structurally diversi?ed Locus I as Type III.
The three groups of archaeal Hsp70(DnaK)se-quences did not cluster together but tended to asso-ciate with separate bacterial groups.However,these associations were characterized by low bootstrap support,whose magnitude depended on the collec-tion of sequences compared (Table 1).The strongest
association was observed between the DnaK se-quences from Methanosarcinales (Group 1)and Firmicutes,with a bootstrap support of 72%in the complete tree of 196eukaryotic and prokaryotic se-quences and of 66%in the prokaryotes-only tree (NJ method).However,the support for this association was much lower (￡40%)for all other trees.The po-sition in the evolutionary trees of the sequences from Thermoplasmatales and M.thermoautotrophicus (Group 2)was variable.Highest support values (55–57%)were obtained for the association with the se-quence from Thermotoga in the ML trees obtained from consensus sequences,but with much lower support (￡30%),and sometimes including the se-quence from Aquifex for other trees.Halobacteriales (Group 3)associated with Actinobacteria with bootstrap support of 35–41%in the NJ trees with prokaryotic or consensus sequences,but in all other trees they joined di?erent clusters with much lower support (￡27%)(Table 1).We concluded that,as in previous analyses (Gribaldo et al.1999),bootstrap analysis does not signi?cantly support the prevailing associations observed in phylogenetic trees of archa-eal and bacterial Hsp70(DnaK)sequences.Moreover,the results were not stable on the choice of tree-reconstruction method and on the selection of data.Amino Acid Composition of DnaK Sequences The three groups of archaeal DnaK sequences asso-ciated with bacterial sequences revealed by our phy-logenetic analyses include sequences translated either from genes of high G+C content (Halobacteriales-Actinomycetes association)and low G+C content (Methanosarcinales-Firmicutes association)or from thermophilic organisms (association of Thermoplas-matales and M.thermoautotrophicus with Aqui?cales and Thermotogales).It is therefore possible that these associations are a consequence of the biases in amino acid usage characteristic of organisms with these genomic or environmental preferences.To determine if the expected compositional biases of the sequences from these organisms extended to the conserved (aligned)regions of DnaK,we characterized all aligned positions of their DnaK sequences by amino acid content,distinguishing (i)content in amino acids encoded by strong bases (G+C-rich codons:Ala,Arg,Gly,Pro),encoded by weakbases (A+T-rich codons:Ile,Lys,Asn,Tyr,Phe),and others;and (ii)content in amino acids overrepresented in thermo-philic organisms (Glu,Gly,Arg,Val,Tyr),in meso-philic organisms (Cys,His,Asn,Gln,Ser,Thr),and others.By using these two three-letter alphabets we determined the similarity in amino acid composition of all sequence groups identi?ed by our phylogenetic analyses (see Materials and Methods).The clusters obtained are shown in Figs.4A (based on
G+C
Fig.2.Phylogenetic tree (neighbor-joining algorithm)of DnaK protein sequences from bacterial,archaeal (prokaryotes),and eukaryotic (eukaryotes)organisms.Similarities of 198sequences were derived from an alignment of 464positions obtained with the multiple alignment procedure Clustal-W (Thompson et al.1994).Pairwise distances were derived assuming gamma-distributed mutational rates with parameter a =0.89.Bootstrap values from 1000replicates are shown for relevant branches when ?500.See Table 1for other relevant bootstrap values.The scale bar repre-sents the indicated number of substitutions per position for a unit branch
length.
Fig.3.Phylogenetic tree (neighbor-joining algorithm)of DnaK protein sequences as in Fig.2,but including only prokaryotic se-quences (98sequences).Pairwise evolutionary distances were de-rived from a protein alignment of 521positions obtained with Clustal-W (Thomson et al.1994)and gamma-distributed muta-tional rates with parameter a =0.79.Bootstrap values from 1000replicates are shown for relevant branches when ?500.See Table 1for other relevant bootstrap values.The scale bar represents the indicated number of substitutions per position for a unit branch length.
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content)and 4B (based on the thermophilic vs.me-sophilic amino acid preferences).Among the groups of archaeal and bacterial DnaK sequences observed in our phylogenetic trees,similarity in amino acid usage did not explain the grouping of Methanos-arcinales with Firmicutes or the grouping of Ther-moplasmatales and M.thermoautotrophicus with Aqui?cales and Thermotogales.However,we ob-served,using both alphabets (Figs.4A and B),the association of the sequences from Halobacteriales with those from Actinobacteriales.This result sug-gests that common biases in amino acid usages may be su?cient to explain the weakassociation of these sequences observed in phylogenetic trees.Structural Features of Bacterial dnaK Loci
For further information on the possible relations of archaeal dnaK loci,we analyzed the structural fea-tures (gene order and separation)in dnaK loci from 124bacterial species classi?ed in 17di?erent lineages.Summary results of this analysis are shown in Table 2(a complete list of gene positions and gene separa-tions for each locus is given in Supplemental Mate-rial,Table S2,online).Bacterial loci containing all three genes (‘‘complete groups’’in Table 2)were most commonly characterized by the arrangement grpE -dnaK -dnaJ .This arrangement was observed among Proteobacteria,Spirochaetes,Chloro?exi,Cyanobacteria,Fusobacteria,and Firmicutes.Vari-ations of the same arrangement were observed in Fusobacterium ,in some Firmicutes and in some Betaproteobacteria,where one gene was inserted be-tween dnaK and dnaJ ,and among a -,b -,and c -pro-teobacteria,where one or more genes were inserted between grpE and dnaK .Incomplete groups with the arrangement grpE -dnaK or dnaK -dnaJ ,possibly remnants of the same complete arrangement,were also observed in other loci from Firmicutes,Cyano-bacteria,Chlamydiales,and a -,b -,c -,d -,and -pro-
teobacteria,where two of the three genes were encoded either next to each other or separated by one or a few genes.The arrangement grpE-dnaK-dnaJ ,however,was not observed in Actinobacteria or in the Deinococcus-Thermus group,which were instead characterized by the gene arrangement dnaK -grpE -dnaJ .Partial groups likely to re?ect the same arrangement were observed in certain actinobacterial loci,which encoded the close pair dnaK -grpE and,separately,dnaJ .Bacteroidetes,Chlorobium ,and Thermotoga encoded the group grpE -dnaJ and,sep-arately,dnaK ,which could have derived from either one of the complete arrangements by means of dif-ferent evolutionary processes.No groups were ob-served in Aquifex aeolicus ,where the three genes were encoded separately from each other.The wide dis-tribution in di?erent bacterial lineages of the ordering grpE -dnaK -dnaJ suggests that this represents the structure of the archetypical bacterial locus.This structure appears to have been repeatedly modi?ed in di?erent lineages by transposition of one or more genes,by duplications,and by occasional insertion of other genes.A rearrangement characterized the evo-lution of the locus in Actinobacteria and in the Deinococcus-Thermus group.
Comparison of the dnaK Bacterial Loci with Archaeal Locus I
In view of the sequence relations of archaeal DnaK proteins with those of Firmicutes,Actinobacteria,and Thermotogales (sometimes Aqui?cales)sug-gested by evolutionary tree reconstructions,it was interesting to consider how the structural features of the dnaK loci in these bacterial groups compared to those of the corresponding archaeal loci.We have observed that among 23genomes of Firmicutes,by far the most frequent gene order was 5￠-grpE-dnaK-dnaJ-3￠(Table 2)as in Methanosarcinales Locus I Type I.Moreover,the distribution of intergenic
dis-
Fig.4.Clustering of bacterial and
archaeal groups based on the amino acid contents of their DnaK sequences in aligned positions.Thermoplasmatales also include the sequence from M.thermoautotrophicus .The scale bar represents 2%di?erence in amino acid type usage calculated as in Materials and Methods.A Clusters
obtained classifying amino acids based on the G+C content of their codons.B Clusters obtained classifying amino acids based on preferences in thermophilic or mesophilic organisms.
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tances observed in Firmicutes between grpE and dnaK(range,17–479bp)and between dnaK and dnaJ (range,57–759bp)(Table3)overlapped those ob-served in Methanosarcinales Locus I Type I,which showed ranges of78–431and66–129bp,respectively (Fig.1and Table2).The resemblance of the struc-tural features of the dnaK loci in the two groups are consistent with the hypothesis of lateral gene transfer of the dnaK locus between Firmicutes and Meth-anosarcinales.However,similar structural features were also observed in bacterial groups as diverse as Proteobacteria,Spirochaetes,Cloro?exi,and Cy-anobacteria,suggesting that they were characteristic of the primordial bacterial locus.
In contrast,the gene order and intergenic lengths observed in Thermotoga maritima(Thermotogales) and Aquifex aeolicus(Aqui?cales)were di?erent from those observed in Thermoplasmatales and M.thermoautotrophicus,in which the three genes occur in the order5￠-grpE-dnaK-dnaJ-3￠(Locus I Type II;Fig.2).In T.maritima,grpE and dnaJ were instead close to one another,separated by6 bp(Table2),and dnaK was encoded in a distant locus.In A.aeolicus,all three genes were in sepa-
Table2.Structural organization of the dnaK locus in bacterial and archaeal phyla
Phylum a Ng b Complete groups Nc c Other groups d Ni e a-Proteobacteria13grpE[X1)2]f dnaK[214/245]dnaJ2dnaK[90/274]dnaJ10 b-Proteobacteria8grpE[61]dnaK[203]dnaJ1dnaK[134]dnaJ1
grpE[X]dnaK[100/266]dnaJ5
grpE[114]dnaK[X]dnaJ1
c-Proteobacteria30grpE[68/263]dnaK[88/325]dnaJ10grpE[119]dnaK1
grpE[X1)6]dnaK[142/209]dnaJ4dnaK[50/226]dnaJ15 d-Proteobacteria5grpE[81]dnaK[112]dnaJ1grpE[5/218]dnaK3
dnaJ[18/259]dnaK2
dnaJ[X]dnaK2
grpE[11]dnaJ1 Spirochaetes5grpE[20/115]dnaK[)1/292]dnaJ5—g—Chloro?exi1grpE[35]dnaK[152]dnaJ1——Cyanobacteria6grpE[282]dnaK[625]dnaJ1grpE[233]dnaK1
grpE[)1/45]dnaJ2
dnaK[)17/108]dnaJ4
dnaK[X2]dnaJ1
dnaK[193]dnaJ[X]dnaJ1 Fusobacteria1grpE[30]dnaK[X]dnaJ1——Firmicutes23grpE[17/479]dnaK[57/759]dnaJ20grpE[)13]dnaK1
grpE[57/59]dnaK[X]dnaJ2grpE[2]dnaK[14]dnaK1 Chlamydiales6——grpE[15/30]dnaK6 e-Proteobacteria4——grpE[21/29]dnaK4 Bacteroidetes2——grpE[42/51]dnaJ2 Actinobacteria15dnaK[)4/86]grpE[)1/204]dnaJ13dnaK[)4/24]grpE3 Deinococcus-Thermus2dnaK[81]grpE[X]dnaJ1——
dnaK[55]grpE[3]dnaJ[)14]dnaJ1
Chlorobi1——grpE[45]dnaJ1 Thermotogales1——grpE[6]dnaJ1 Aqui?cales1————Methanosarcinales6grpE[78/431]dnaK[66/129]dnaJ6dnaK[116/176]dnaJ2 Thermoplasmatales4grpE[)23/6]dnaK[8/16]dnaJ4——Methanobacteriales1grpE[14]dnaK[150]dnaJ1——Halobacteriales3grpE[X]dnaK[76/142]dnaJ2——
grpE[165]dnaK[X4]dnaJ1
a Archaeal species group names in boldface.
b Number of genomes examined.
c Number of times the complete gene group was observed.
d In th
e instances shown,the genomes contained gene groups that were incomplete,i.e.,with one o
f the three genes missing.
e Number o
f times the incomplete gene group was observed.
f When one or more other genes are inserted between the two genes shown,this is indicated by X when one gene is inserted or by X
1)6
when, for example,from one to six genes are observed inserted in di?erent instances.Figures in brackets represent the number of nucleotides (length of the intergenic region)separating the contiguous genes shown.If there are two?gures within the brackets they indicate the lower and upper values(range)of the number of nucleotides separating two contiguous genes,when the gene group was observed in di?erent instances.A minus sign indicates that the two genes are overlapping by the indicated number of nucleotides.
g A dash signi?es that no instances of the indicated gene group type(complete or not)were observed.In the case of Aqui?cales no gene groups were observed;the genes were found separated,far apart from each other in the genome.
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rate loci(Supplemental Material,Table S2,online). Similarly,the gene order observed in Actinobacteria did not re?ect the structural features observed among Halobacteriales(Locus I Type III;Fig.3), in which the gene order was5￠-grpE-NN-dnaK-dnaJ-3￠(NN denotes an unidenti?ed gene interposed between grpE and dnaK).In fact,as we have seen (Table2),the genes of Actinobacteria were most frequently organized in the order5￠-dnaK-grpE-dnaJ-3￠,with dnaK and grpE near one another. Discussion
Locus I T ypes:Evolutionary and Functional Implications
Many archaea do not have the stress gene dnaK that encodes the molecular chaperone DnaK,which is highly conserved in sequence and is always present in bacteria and eukaryotes(Macario et al.2004).We found that in the archaeal species in which dnaK does occur,this gene is in a locus,termed dnaK Locus I, typically along with the genes grpE and dnaJ, encoding chaperones known to interact and cooper-ate with DnaK in protein folding(Georgopoulos et al.1990;Bukau et al.2000;Zmijewski et al.2004).The three genes were organized as follows:5￠-grpE-dnaK-dnaJ-3￠,except in Halobacterium sp.NRC-1 and H.mediterranei,in which a gene of unknown function was predicted between grpE and dnaK (Figs.1A–C).Furthermore,in H.marismortui,the locus encoded grpE and dnaK adjacent to one an-other but dnaJ separated from dnaK by four pre-dicted genes.These?ndings indicate that haloarchaea are quite diverse with respect to the dnaK-locus genes.
Three Locus I types were identi?ed and named I, II,and III.These Locus I types di?ered in structural and organizational details,and corresponded to the clusters,termed Groups1,2,and3,of archaeal DnaK proteins revealed by phylogenetic analyses. Locus I Type I occurred in the archaeal organisms whose DnaK proteins formed Group1,and these proteins clustered with the DnaK proteins from Fir-micutes(Gram-positive bacteria with low G+C content in their DNAs),with the occasional addition of the sequence from Fusobacterium;interestingly, this archaeal Group1includes mesophilic,moder-ately thermophilic,and psycrophilic species.
Locus I Type II occurred in the archaeal organ-isms whose DnaK proteins formed Group2,and these proteins clustered with the DnaK proteins from Thermotogales,sometimes in association with Aqu-i?cales.Locus I Type III was found in the archaeal
Table3.Intergenic distances in the dnaK loci of Firmicutes with the typical gene cluster
Intergenic distance between the genes(bp) Species a Strain grpE and dnaK dnaK and dnaJ Bacillus cereus ATCC1098726204
Bacillus licheniformis DSM1323182
Bacillus subtilis Not speci?ed2393
Bacillus thuringiensis97–2726204 Clostridium acetobutylicum ATCC82420125 Clostridum perfringens1356113 Clostridium tetani E886572 Lactobacillus johnsoni NCC5331779 Lacatobacillus plantarum WCFS143101
Listeria innocua Clip1126233142
Listeria monocytogens EGD-e33141 Oceanobacillus iheyensis Not speci?ed34148 Staphylococcus aureus Mu5068135 Stahylococcus epidermidis ATCC1222855144 Streptococus agalactiae Not speci?ed180288 Streptococcus mutans UA159373527 Streptococcus pneumoniae R6479759 Streptococcus pyogenes M1GAS180280 Streptococcus thermophilus CNRZ1066128426 Thermoanaerobacter tengcongensis Not speci?ed1857
Range17–47957–759 Arithmetic mean94211
Median38.5143
a Among the genomes from23species that were examined,20(listed in the table)had the typical cluster5?-grpE-dnaK-dnaJ-3?,whereas the other3had some variation of it:an extra gene between dnaK and dnaJ(Bacillus halodurans C-125and Enterococcus faecalis V538)or dnaJ located at a distant site,i.e.,not clustered with the other two genes in the chromosome(Lactococcus lactis https://www.wendangku.net/doc/eb5443111.html,ctis).The Clostridium acetobutylicum ATCC824genome contains,besides the typical cluster,a second cluster where the grpE gene is followed by two copies of the dnaK gene.
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organisms whose DnaK proteins formed Group3, and these proteins clustered with the DnaK proteins from Actinobacteria(Gram-positive bacteria with high G+C content in their DNAs).All of the organisms with Type III Locus I were extreme halo-philes and showed a diversity in locus organization that was not observed in the other two Locus I Types. This diversity parallels other kinds of variations ob-served in haloarchaeal genomes,suggesting that these genomes are somewhat variable by comparison with other archaeal groups, e.g.,the methanogens(Ng et al.2000;Baliga et al.2004).
In previous work,it was demonstrated that the genes in the triad5￠-grpE-dnaK-dnaJ-3￠in M.mazei S-6are transcribed individually from a di?erent pro-moter for each gene,rather than as an operon from a single promoter as in many bacteria(Clarens et al. 1995;Conway de Macario et al.1995).The structure and organization of the triad in M.mazei S-6are very similar to those in the other Methanosarcina species studied in this work,all of which display Locus I Type I.Therefore,we may predict that in all Methanosar-cina species,and perhaps in all archaea with Locus I of Type I,the same mode of transcription will occur. In fact,putative promoters have been identi?ed up-stream of the three genes in these organisms(Hickey et al.2002).In contrast,there is no room for a typical promoter in the intergenic regions upstream of dnaK and dnaJ in Locus I Type II,found in Thermoplas-matales and in M.thermoautotrophicus.We may then infer that in organisms with a Locus I of Type II,the mode of transcription of the genes in the triad5￠-grpE-dnaK-dnaJ-3￠is di?erent from that of the genes in Locus I Type I.The three genes in Thermoplasmatales could be transcribed as a single operon,and at least grpE and dnaK could be transcribed together in M. thermoautotrophicus.In all these organisms,bearing a Locus I of Type II,the transcription would be com-manded from a single promoter upstream of grpE,as is the case for some bacteria.
The structural features of Locus I Type III(pres-ent in Halobacteriales),in terms of length of inter-genic regions upstream of grpE,dnaK,and dnaJ,and in terms of presence of putative promoters,are sim-ilar to those of Type I.These similarities suggest that the mode of transcription of the genes in Locus I Type III is monocistronic,commanded by a pro-moter for each gene,as has been demonstrated for the genes of Locus I Type I in M.mazeii S-6.
Origin of the Archaeal dnaK Genes
Our phylogenetic analyses revealed three distinct groups of archaeal DnaK sequences,suggesting that, within each of these groups,the dnaK gene derived from a common ancestor through vertical descent. Groups1and3comprised,respectively,sequences from Methanosarcinales and Halobacteriales,and they conformed to recognized taxonomic classi?ca-tions.Group2included all sequences from Ther-moplasmatales and the sequence from M. thermoautotrophicus,indicating that Thermoplasma-tales and Methanobacteriales possess dnaK genes derived from the same ancestor.
In contrast to the internal cohesiveness of the three archaeal dnaK-gene groups(i.e.,Locus I Types I–III) demonstrated by genomic studies and by the phylo-genetic analyses of their protein products,the evo-lutionary relations among these three groups were not evident.The three groups of archaeal DnaK protein sequences did not cluster together to form an archaeal supercluster,as we might expect from the cohesiveness of the archaeal domain as a whole,but, in agreement with previous results(Karlin and Brocchieri1998;Gribaldo et al.1999),they seemed to associate with distinct bacterial groups,suggesting separate events of lateral gene transfer(LGT).
LGT from bacteria has been previously suggested in the evolution of archaeal dnaK genes(Gribaldo et al.1999)and as an important factor in the evolu-tion of archaeal genomes(Aravind et al.1998).The hypothesis that archaeal dnaK genes have been hori-zontally transferred from organisms belonging to separate bacterial clusters is an attractive proposition that may explain the lackof an obvious relation among the three groups of archaeal DnaK proteins and their association with distinct bacterial groups. These associations could also help in predicting the modes of transcriptional regulation for the genes in the various archaeal groups,based on what is known for the bacterial species closest to each group.
Among the three archaeal groups,the association of Methanosarcinales with Firmicutes was the most strongly supported,although with bootstrap values often less than50%(Table1).The structural simi-larities of the loci involving the grpE,dnaK,and dnaJ genes in Firmicutes and in Methanosarcinales(same order and similar intergene spacer lengths)also sug-gested that these genes have been laterally transferred between the two groups.However,we noticed that the structural features common to Firmicutes and Methanosarcinales were also common to many other bacterial lineages,suggesting the possibility that modern-age Firmicutes and Methanosarcinales may have independently inherited the structural organi-zation of their loci from a primitive prokaryotic ancestor.Furthermore,although in M mazei S-6and probably in other Methanosarcinales,each gene of Locus I is controlled by a separate promoter(Clarens et al.1995;Conway de Macario et al.1995),the dnaK locus of Firmicutes is polycistronic,as has been ob-served in B.subtilis(Homuth et al.1997),Listeria monocytogenes(Hanawa et al.2000),and Clostridium acetobutylicum(Narberhaus et al.1992)or predicted
83
in Staphylococcus aureus(Wang et al.2004).This di?erence in functionality of the intergenic regions of the locus in Firmicutes vs.Mathanosarcinales may suggest that their similarity in length could have evolved independently in the two lineages.
The associations between archaeal and bacterial DnaK sequences for loci of Type II(Thermotoplas-matales and M.thermoautotrophicus)and of Type III (Halobacteriales)were tenuously supported by bootstrap analysis and largely depended on the choice of data and clustering method.We have been able to show that in the case of Halobacteriales and Actinobacteria these associations may result from the biases in amino acid usages that characterize genomes of high G+C content(Fig.4).Sequence biases that characterize proteins of thermophilic organisms may also contribute to the clustering of DnaK sequences from Thermoplasmatales and M.thermoautotrophi-cus with Thermotogales(Kreil and Ouzounis2001; Tekaia et al.2002).Besides poor bootstrap support
for the associations between these DnaK sequences, we also found that there was no similarity in the structural organization of the loci between the cor-responding archaeal and bacterial genomes.
It is interesting to consider the LGT hypothesis in view of recent reconstructions of the phylogeny of archaeal organisms,based on16S rRNA and ribo-somal proteins(Forterre et al.2002;Brochier et al. 2005).These phylogenetic relations imply that if ar-chaeal dnaK genes were acquired by three indepen-dent LGT events,the evolutionary history of the gene in Archaea must have consisted of a complex pattern of acquisitions,losses,and substitutions.Among the possible scenarios,the most parsimonious,illustrated in Fig.5,implies a primordial acquisition of a Type II gene(encoding DnaK of Group2),followed by two independent losses of the same gene in the lin-eages of Methanococcales and in the progenitor of Archaeoglobales,Halobacteriales,and Methanos-arcinales.These events must have been followed by the independent acquisition in the Methanosarcinales and Halobacteriales of,respectively,Types I and III genes(encoding DnaK proteins of Groups1and3).
A scenario of vertical descent with lineage-speci?c structural and functional di?erentiation would in-stead imply independent gene loss events in the ar-chaeal lineages(hyperthermophiles?)that do not encode the hsp70locus.
As for the relationships between archaeal and bacterial sequences,inconsistent associations were also observed for DnaK sequences of di?erent major bacterial lineages when comparing di?erent trees (Figs.2and3;also Supplemental Figs.S2–S6).This sort of result is common in reconstructions of bac-terial phylogenies based on protein sequences (Teichmann and Mitchison1999;Brocchieri2001).The di?culty in distinguishing ancestral bacterial relationships has been attributed to the accumulation of multiple mutational events in all variable positions of protein sequences,i.e.,mutational saturation (Philippe and Laurent1998;Brocchieri2001). Somewhat paradoxically,more distantly related ar-chaeal and bacterial protein homologs are more clearly distinguished(Lopez et al.1999),a phenom-enon that can be explained by the characteristic functional di?erentiation speci?c to each phyloge-netic domain that is observed for many homologous genes(e.g.,hsp60or recA genes)between Bacteria and Archaea,but that is not obvious from the anal-ysis of bacterial and archaeal DnaK sequences.
A lackof substantial functional di?erentiation between archaeal and bacterial DnaK sequences may explain the uncertainties in determining their evolu-tionary relationships.However,di?erences in struc-tural features indicate that various transcriptional-regulatory modes,perhaps only slightly di?erent from one another,have evolved in the three types of dnaK Locus I from di?erent archaeal groups.A comparison of the structural and functional features of the dnaK locus in Bacteria and Archaea suggests the possibility that the di?erent types of archaeal dnaK loci extant today derived and independently evolved from a primordial archaeal ancestor(or from a meta-genomic organism;Woese1998). Acknowledgments.We thankYimin Dong,Brian W.Meneghan, and David A.Gross for their help with phylogenetic and genomic analyses and the San Francisco Foundation for funding to A.J.L.M.and E.C.deM.A.R.S.wishes to thankProf.Sandhya S. Visweswariah for useful discussions and the Indian Institute of Science,Bangalore,for scholarship.L.B.was supported by NIH Grant2RO1GM010452and wishes to thankProf.Sam Karlin for useful
discussions.
Fig.5.Schematic phylogenetic tree of archaeal organisms and putative pattern of dnaK gene loss/acquisition.The phylogenetic relationships of archaeal organisms are based on16S rRNA and ribosomal protein sequences(Forterre et al.2002;Brochier et al. 2005).One of the possible scenarios of dnaK gene loss/acquisitions in various archaeal lineages is shown.Circled I,II,and III repre-sent independent insertions of Locus I Type I,Type II,and Type III genes in Archaea by lateral gene transfer.Crossed-out circles represent losses of the corresponding loci.
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2020最新MACH软件简单安装设置
MACH3软件简单安装设置 安装软件一步一步默认安装 软件驱动一定要勾起。不然会没有输出，或黑屏。 安装后记得先不要运行软件，一定要重启电脑。
桌面我的电脑，右键-管理-计算机管理-设备管理器-MACH3驱动要显示正常，无“！”和“？”软件才能正常使用，如无安装上MACH3驱动或驱动上显示有“！”并口没信号输出。 桌面有快捷图标一个是洗一个是车我们运行洗 首先设置单位：左上角“设置”里的“公/英制选择”选公制毫米 设置—端口/针脚，端口设置和轴向选择：如果你只有一个并口就选这个如果是步进选25000HZ 是司服可以选高点，注意该打√和要填数值的都和图一样设置，设置好后点应用
设置—端口/针脚，电机输出：设置接口脉冲及方向输出，这个定义要根据接口版填写，使能勾起，方向端口勾或不勾可以改变电机方向，脉冲要根据接口版勾或不勾，后面两个1 是第一并口，设置好后点应用。 设置—端口/针脚，输入信号：如果装了限位限位有超限和原点，可以用一个端口脚并联，因为一个并口就5个输入，设定后点应用。
设置—端口/针脚，输入信号：Estop如果软件急停不停闪这要设置一下，P10并口10脚本外部急停脚，HotKey为急停快捷键设定点一下弹出窗口 此时按一下键盘上你要设定的急停快捷按键即设定后点应用 设置—端口/针脚，输入信号：外部按钮设置这是设置13端口可作对刀信号，HotKey为快 捷键设定点一下弹出窗口此时按一下键盘上你要设定的急停快捷按键即设定后点应用
设置—端口/针脚输出，输出信号：14为使能控制信号同时也是外置手控制的切换，14信号设好后软件急停下可以用外置手动控制，软件待机或运行时关闭外置手运控制 Output #1、为主轴继电器开关设置，Port # 设为1（这里的1是指第一个并口）Pin Number 设为1（这里的1为并口定义第一脚做主轴控制信号） 设置—端口/针脚输出，主轴设置：启用主轴继电器控制时要把（禁用主轴继电器）框里的勾去掉
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电脑主板接口图解
主板内存插槽、扩展插槽及磁盘接口： DDR2内存插槽 DDR3内存插槽 内存规范也在不断升级，从早期的SDRAM到DDR SDRAM，发展到现在的DDR2与DDR3，每次升级接口都会有所改变，当然这种改变在外型上不容易发现，如上图第一副为DDR2，第二幅为DDR3，在外观上的区别主要是防呆接口的位置，很明显，DDR2与DDR3是不能兼容的，因为根本就插不下。内存槽有不同的颜色区分，如果要组建双通道，您必须使用同样颜色的内存插槽。
目前，DDR3正在逐渐替代DDR2的主流地位，在这新旧接替的时候，有一些主板厂商也推出了Combo主板，兼有DDR2和DDR3插槽。 主板的扩展接口，上图中蓝色的为PCI-E X16接口，目前主流的显卡都使用该接口。白色长槽为传统的PCI接口，也是一个非常经典的接口了，拥有10多年的历史，接如电视卡之类的各种各样的设备。最短的接口为PCI-E X1接口，对于普通用户来说，基于该接口的设备还不多，常见的有外置声卡。
有些主板还会提供迷你PCI-E接口，用于接无线网卡等设备 SATA2与IDE接口
横向设计的IDE接口，只是为了方便理线和插拔 SATA与IDE是存储器接口，也就是传统的硬盘与光驱的接口。现在主流的Intel主板都不提供原生的IDE接口支持，但主板厂商为照顾老用户，通过第三方芯片提供支持。新装机的用户不必考虑IDE设备了，硬盘与光驱都有SATA版本，能提供更高的性能。 SATA3接口 SATA已经成为主流的接口，取代了传统的IDE，目前主流的规范还是SATA 3.0Gb/s，但已有很多高端主板开始提供最新的SATA3接口，速度达到6.0Gb/s。如上图，SATA3接口用白色与SATA2接口区分。 主板其他内部接口介绍：
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大金空调机远程监控板说明书 RX/B TX/A GND VIN2 VIN1 P2 P1 P2 P1 图一监控板外形及端口示意图 3、通信口连接： ①控制器的通信接口有RS232和RS485两种，用户可以根据实际 需要通过跳线J4、J5自行选择其中一种使用； ②连接方法： 1、VIN2和VIN1接空调室内板的X35插线端子，没有极性。（也可外部供电，8—15V，AC、DC均可） 2、P1、P2接空调机室内外板通信端子座，没有极性。（本控制器为一拖二型，可以接两台空调，地址为连续的两个地址来区分，如控制器板地址设置1时，另一台的地址则为2，如控制器板地址设置2时，另一台的地址则为3，依次类推） 3、RS485通信： RX/B为RS485的（－），TX/A为RS485的（＋） 第2 页共4页
PCB电路板PCB布线知识
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博客首页┆播客┆圈子┆美图┆专栏┆搜 索┆帮助 猪猪的BLOG http://blog.sina../homeofyanyan复 制>收藏本页添加为友情链接 博客首页 个人首页┆管理博客┆我的文章┆我的 相册┆我的圈子┆我的播客┆我的好友┆ 给我留言 PROTEL元件封装
2007-06-2316:38:49 大中小 电阻AXIAL0.30.4 三极管TO-92AB 电容RAD0.10.2 发光二极管DZODE0.1 单排针SIP+脚数 双排针DIP+脚数 电解电容RB.1.2。。。。。。。} 电阻AXIAL 无极性电容RAD 电解电容RB- 电位器VR 二极管DIODE 三极管TO 电源稳压块78和79系列TO－126H和TO-126V 场效应管和三极管一样 整流桥D－44D－37D－46
单排多针插座CONSIP 双列直插元件DIP 晶振XTAL1 电阻：RES1，RES2，RES3，RES4；封装属性为axial系列 无极性电容：cap;封装属性为RAD-0.1到rad-0.4 电解电容：electroi;封装属性为rb.2/.4到rb.5/1.0 电位器：pot1,pot2；封装属性为vr-1到vr-5 二极管：封装属性为diode-0.4(小功率）diode-0.7(大功率） 三极管：常见的封装属性为to-18（普通三极管）to-22(大功率三极管）to-3(大功率达林 顿管） 电源稳压块有78和79系列；78系列如7805，7812，7820等 79系列有7905，7912，7920等 常见的封装属性有to126h和to126v
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目录 前言 .......................................... 错误!未定义书签。第一章机床特点及性能参数. (2) 1.1机床特点 (2) 4.1 准备工作 4.2 上电试运行 (8) 第五章主轴系统 (9) 5.1 简介 (9) 5.2 主轴系统的机构及调整 (10)
5.2.1 皮带张紧 (10) 5.2.2 主轴调整 (11) 5.3 动力卡盘 (11) 第六章刀架系统 (11) 第十一章机床电气系统 (14) 11.1主要设备简要 (15) 11.2 操作过程： (15) 11.3 安全保护装置： (15)
11.4 维修： (15) 第十二章维护、保养及故障排除 (18)
前言 欢迎您购买我厂产品，成为我厂的用户。 本说明所描述的是您选用的我厂YCK-6032/6036标准型全功能数控车床。该车床结构紧凑，自动化程度高，是一种经济型自动化加工设备，主要用于批量加工各种轴类、套类及盘类零件的外圆、内孔、切槽，尤其适用轴承行业轴承套圈等多工序零件加工。
第一章机床特点及性能参数 1.1机床特点 YCK-6032/6036全功能数控车床是顺应市场要求向用户推荐的优秀产品，该机性能优异，各项指标均达国际水平，具有较高的性价比，可替代同类进口产品。 YCK-6032/6036整机布局紧凑合理，其高转速、高精度和高刚性，为用户在使用中提 本机标准配置为排刀架，刚性好，可靠性高，故障率低，重复定位精度为 0.007mm，相邻刀位移动时间为0.3秒，车、镗、钻、扩、铰等工具可同时安装使用。 另外，本机可选配八工位、十工位、十二工位液压转盘刀塔。 本机进给系统全部由伺服电机（可选配步进电机）直连驱动，刚性、动态特性好，系统的最小设定单位为0.001mm，快速移动速度为X轴15m/min，Z轴15m/min，
FANUC机床使用说明书
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目錄 第一章電氣規格 第二章機床使用前注意事項 電氣規範 第一次啟動前注意事項 機床用電安全 機床操作注意事項 機床接地及注意事項 開機關機順序 開機操作順序 關機操作順序 機械原點複歸 緊急停止及恢復 第三章操作面板 機械操作面板外觀圖 機械操作面板功能說明 . 電源及緊急停止區 . 模式選擇區 . 軸的移動以及速度倍率選擇區 . 主軸控制區 . 自動操作功能區 . 周邊功能區
. 警示燈區 . 指示燈區第四章功能代碼表 第五章異警排除 附錄列表 附錄值設定一覽表 附錄定時器設置一覽表附錄計數器設置一覽表
第一章電氣規格 第二章機床使用前注意事項 電氣規範 本機床目的是用於金屬切削。例如銅、鐵、不銹鋼、鋼、鋁以及鋁合金屬。其他金屬或用途是不允許的。而且不適合使用在易燃物質或硬脆材質如鎂、陶瓷、木頭、玻璃和有毒物質等。假如有任何標準問題，為了防止人員的安全和保障貴公司的權益，請和本公司聯絡。 第一次啟動前注意事項 ●操作前請仔細閱讀本書和數控系統的使用手冊 ●由於經過運輸過程中的顛簸，拆箱後請先檢查三向的運輸固 定裝置是否完好，檢查主軸箱與配重錘之間的連接件是否牢固可靠。 ●在機床運行之前，必須檢查三向及主軸箱和配重錘的運輸固 定架和固定螺釘是否已全部拆除。 ●首次啟動機床或停用較長時間後，再次啟動機床時，打開電 源後，應等待分鐘，待機床充分潤滑後，再操作機床。 ●在機床首次使用前，必須將主軸打刀用增壓缸的油杯注滿液 壓油，並排除缸體中的氣體，以確保打刀的可靠性及打刀力，從而避免損傷機床及人員。
MACH3常用设置_教学
Mach3Mill，铣床主界面。 界面上的内容非常多，乍一看感觉很复杂，这也是我第一次接触Mach家族软件的第一印象。 经过一段时间的使用和研究后发现，实际上只要进行简单的几个设置之后就可以初步的运行了。待我慢慢道来。 打开软件后的第一步就要确定用于计算机床进给系统所使用的长度单位是公制还是英制，也就是毫米还是英寸。 打开Config菜单，选择第一项Select Native Units。
随后会弹出一个提示窗口，不用理睬直接点击OK。 (提示内容是告诉用户这里的长度单位的确定与G程序中使用的长度单位没有关系，这里确定机床步进(伺服)电机，在进给运动时所使用的长度单位。) 之后就会出现单位定义窗口了。 我使用了mm‘s，也就是毫米，因为我使用的丝杠是2.5mm导程(螺距)的，是公制的的丝杠。所以这样可以方便的计算出步进电机的转速，而不存在单位换
算出现的误差。 按下ok后即可。 第二步，开始设定你的Mach接口，定义并口引脚功能。这也是Mach中设定最关键的部分，机床是否能够正常的运行就靠这里的设定。 Config菜单-Ports and Pins项 弹出Ports & Pins对话框，此对话框内有多个标签窗口。首先看到的是Port Setup and Axis Selection标签窗口。
窗口中有以下选项是我们要关注的，Port #1中定义了PC主板上唯一的一个并口地址，这个地址在主板BIOS中已经定义一般为默认，无需要更改，Port Enable打勾有效。Port #2定义如果主板上有第二个并口，则定义了第二个并口的地址。 Kernel Speed核心频率定义了mach系统的最高运行速度，决定了机床进给速度的极限，无论是步进电机还是伺服电机最高脉冲频率决定了其转速，所以Kernel Speed的频率限制其最大脉冲频率。 一般步进电机的转速比较低，极限转速大约700转，标准脉冲是每转200个，如果使用细分驱动器达到8细分的话就是1600个脉冲转一圈。每分钟 700×1600/60秒=18666.66HZ/秒。如果步进马达要达到700转就要使用高于18666.33HZ/秒的核心频率，应该选择系统中的25000HZ，就可以了。 Port Setup and Axis Selection标签窗口其他选项可暂时不用设定。
线路板型号命名规则
一、目的及范围 统一规划产品开发中所涉及电路板型号命名，提供和识别产品具体信息内容及相关文档的可控性，便于操作和统一管理，特此规范与说明。 作用范围包括环氧、铝基、瓷基、柔性、纸基等通用型线路板。 二、适用性 适用于xxxxxx 硬件开发部。 三、公司产品开发思路 当前所发布的产品和公司的业务发展方向----向智能感知、物联网方向发展，因此，为 保证产品开发进度，采用模块化产品开发模式，不同模块组合构成柔性的、可变的、多样化的产品，从而尽量缩短开发时间，同时减少商务、生产、测试的物流流转时间，为争取最快的上市时间提供保障。实现“以不变（模块系列）应多变（用户需求）的产品开发模式。 模块化设计的基本方法： 新产品=不变部分（通用模块）+准通用部分（改型模块）+专用部分（新功能模块） 从公司当前业务发展和及方向看，通用模块主要有（以后有新的需求再增加）： A ：基于视频分析应用通用模块； B ：基于物联网应用的通用模块； C ：基于逻辑控制的通用模块； D ：基于数据交换的通用模块。 因此线路板的命名分为通用模块线路板命名规则和专用功能接口线路板命名规则。 1、通用模块线路板命名规则 商标“HFC ” 业务应用类型 特征信息 附属信息 版本信息
商标信息：固定为“HFC ”； 业务应用类型（最多3位）： 基于视频分析应用通用模块：标识“A ”； 基于物联网应用的通用模块：标识“M2M ”； 基于逻辑运算控制类通用模块：标识“ LOC ”； 基于数据交换的通用模块：标识为“SW ”。 *若后续有补充，可进行增添。 特征信息（最多3位）： 主要描述通用模块关键特征，利于区分相同业务应用类型模块之间差异。例如：交换机 有5以太网，则此位标识“5”，有8口，则此位标识“8”。如果没有，默认用“n ”标识。 附属信息（最多4位，可数值也可文字） 主要表述核心芯片的信息，诸如，A8板采用TI Davinic DM6446芯片，则在附属信息中“6446”用于标识； 版本信息（2位数值） 该标识位表示线路板的版本，用括号内数值代表，默认第一版用“(10)”（以版本号右 移一位作为版本标识），若更改线路板相关内容，即改版打样，数值相应增加，如改过一次大的，一次局部布局，并打样，最新版本为“(21)”。 2、专用部分线路板命名规则 在此之前的产品没有按此规则命名的，在改版后必须按以下命名规则执行。 商标“HFC ” 产品类型 用途信息 附属信息 版本信息 商标信息：固定为“HFC ”；
车床操作说明书
车床操作说明书 一. 操作注意事项 1. 起动时,为安全应查看起动杆(18)是否在停止位置,车床启动时应使主轴空转1~2分钟,使润滑油散布各处,等车床运转正常后方能进行工作; 2. 控制开关(1)向右转,电源指示灯(2)亮,此时即表示可依起动杆予以操作,起动杆(10)由中心移到左倾之状态为正转,右倾状态为反转,而中央为停止; 3. 车前电极时,移动横向手动轮(14),使刀尖(18)与工件(17)待加工面接触,数显器Y轴归零,车削到外圆见光,然后用分厘米卡后再车削,车削到所需之尺寸.在车削过程中进刀量0.15mm,到最后进刀量0.02~0.05mm之间;(见图A) 4. 工作需变速时,必须先停车,方可移动主轴变速杆(5),需要高低速转换必须先停车,方能移动主轴高低速变换杆(8); 5. 车刀在车削工件过程中,如果车刀磨损应及时刃磨,用钝刃车刀切削会增加车床负荷,甚至损坏机床; 6. 选择正确的刀具切削不同的材质,装夹刀具时,刀杆伸出长度是刀具厚度的1~1.5倍,刀具一定要对正工件中心; 7. 车牙时,打开后部盖(25),依切螺丝装更换齿轮,操作押送正反转转旋钮(6)公英制变换杆旋扭及押送变速杆(11),九段排檔(7)选择后锁紧杆固定.选择厘米牙切削时制牙切削或自动押送(11)做螺牙押送时,将选择把手(15)量于中央位置,操作切牙杆(13); 8. 车削中应适当加切削液,以减轻刀具磨损; 9. 根据刀和材质选择不同的速度; 二. 操作时必须提高执行纪律的自觉性,遵守规章制度 1. 工作时应穿工作服、工鞋、戴袖套.工作时,头不应靠得工件太近,以防止切屑溅入眼内,车削崩碎状切削工件时,必须戴防护眼镜; 2. 工作时,必须集中精力,身体和手不能靠近正在旋转的工件或车床部件; 3. 工件和车刀必须装夹牢固,以防止飞出发生事故; 4. 不准用手去剎住转动的卡盘; 车床开动时,不能测量工件,也不能用手去摸工件表面; 5. 不准用手去剎住转动的卡盘;加工中不能用手清除铁屑,应用专用的钩子清除,绝对不允许用手直接清除; 6. 在运转中车床不可变速,不可动(5)(8),变速中转动卡盘看齿轮是否完全啮合; 7. 加工中不能用手清除铁屑,应用专用的钩子清除,绝对不允许用手直接清除; 8.加工工件过程中,应多次测量,以保证工件质量要求. 模具部车床组新进员工培训计划 一.思想观念教育及课内管理规定教育(课内管理数据,含制作流程教育) 二.掌握第三视角法,了解课内模具图面之识图方法. 三.熟悉三角函数之间的换算及运用. 四.了解模具各组件的名称及作用(同模具课模具组件教育数据) 五.了解常用材料特性及用途 六.车床基础知识教育 6.1车床加工原理; 6.2车床部件名称及其作用和百分表使用及保养; 6.3车床保养与维护; 6.4 车床操作程序及注意事项, 6.5车床加工工件标准.(同QC检测标准) 及加工内容之认识;
Mach3教程
安装培训教程声明 本雕刻机作为网络交流的个人作品。成品及半成品及套件并非严格意义上的商品使用者需具备相关知识凡是涉及机械、电子、计算机的设备都有可能因使用不当或病毒、与其它软件兼容原因等造成故障此故障可能造成一定的危险及经济损失本人不对直接及间接损失承担相应责任。有关软件版权本机器所涉及的相关软件均来自互联网原作者享有版权作为学习了解之用请及时删除并购买授权软件使用没有授权的软件造成一切损失及法律问题由使用者自行承担。有关培训范围本人只对CNC雕刻机承担相应的责任货款只是设备本身的价格未包含任何软件及软件培训费用货到后用户在手册指导或通过网络在作者指导下设备调试成功即确认作者的工作完成本设备使用过程中所涉及到的所有软件不在作者的培训责任之内作者只能给予适当知道及在自己则能力之内给予答疑解惑网络时代请广大玩家尽量利用网络工具求助交流 设备及软件的安装及设置警告变频主轴属于精密高速专业主轴变频是设置非常专业设置不当将造成变频器和主轴电机的损毁用户不要私自更改变频器设置不要拆解主轴电机和变频器变频器内部有高压可能对您造成伤害变频器的频率很高如果设备接地不合格可能对系统造成干扰不能正常工作。数控雕刻机是依靠相关软件控制工作的设备上的一些安全触发装置也是依靠正确的软件设置才能正常运行在没有完全确认设置正确的情况下冒然装刀试机可能都设备造成永久的损伤本设备采用计算机并行接口和PC连接控制软件MACH3通过并口端口控制雕刻机各轴按照指令运行WINDOWS请用sp2版本其他版本可能出问题提示并口打印口要求工作在EPP模式任何其它模式可能造成雕刻机不能正常运行有关EPP模式的设置应在计算机主板BIOS中进行各个厂家的设置方法不尽相同请参阅计算机的说明书进行设置。警告控制用的PC应该是台专用的并尽可能不要按装其它应用软件警告部分PC没有自带的并行口玩家需另行购买PCI插槽的并口扩展卡任何市售的USB-并口打印口的设备都不能使本设备正常运行。本人并不建议用笔记本电脑控制本设备如果一定要用请查看笔记本电脑的手册关掉有关电源管理等相关功能一、控制软件MACH3的安装警告在软件的安装及设置过程中请不要开启雕刻机电源以免产生误动作发成意外 1、在随机光盘“MACH3 2.63”目录中打开文件夹“MACH3” 2、运行“MACH3 R2.63.EXE”开始安装全部默认点击“NEXT”直到安装完成3、将“覆盖安装目录”中的全部文件覆盖到软件的安装路径默认状态下是C:\MACH3,确认覆盖。4、重新启动您的PC 5、正确安装了软件后在系统的设备管理中应该能看到相应的标示右键点击桌面图标“我的电脑”----“属性”----“硬件”----“设备管理”----可以在列表中看“Mach3 Driver”如果没有应该重新安装软件重新安装之前应该卸载原来的并手工删除其目录。二、MACH3的设置重新启动PC后桌面多了几个新的图标我们能用到的就是“Mach3Mill”双击之进入如下的控制界面
旋压机使用说明书(新)
CT系列卧式旋压机 使用说明书 （机械部分）
前言 十分感谢您选择了我们制造的复合旋压机床。 正确的使用、维护机床可以为您创造更多更好的财富，也能够使机床保持长久、稳定的精度和寿命。另外，对操作者的人身安全和机床和机床的使用安全也有了可靠的保障。因此，在开始使用本机前，请您务必首先阅读和理解本使用说明书的各个章节，特别是安全方面的章节。 关于本机床操作方面及NC编程方面的知识，请详看本《使用说明书》的电气部分。 由于在生产过程中，对机床某些地方进行小的变动是不可避免的，因此当本说明书与机床间出现小的差别时，恕不通知。
1 安全 当您使用CT-250M/450M复合旋压机床时，请认真阅读以下各项安全注意点，若有疏忽，轻者可能降低切削精度，重者可能引起严重的身体伤害或更加不堪设想的后果。 1.1安装程序 1.1.1安装前准备工作 （1）电源：本机床需要的电源供应容量，规格如下： (2)气源：气压用以控制尾轴、料架控制、切边刀控制及工件吹气等，其 设计压力为5bar，因此气源的压力值应稳定的保持在6bar以上，本 机用量为300l/min。 （3）润滑油箱的油量一定要充足，其容量为1.8L。 1.1.2 安装环境 1. 环境条件 通常机床应安装在以下条件的环境内： （1）电源电压：85%到110%变化波动范围内 （2）频率：±2Hz变化波动范围内 （3）室温：0℃到45℃范围内 （4）相对湿度：＜90%（温度变化不应引起冷凝现象） （5）空气：避免高度灰尘，酸腐蚀气体和盐雾 （6）机床不应安装在阳光可引起环境温度变化的位置和安装在有 异常振动的环境内。 2.机床应与其他机床分开放置，不可距离大近。 3.应有足够的维修空间。安装时，机床的门及控制器应不影响或阻碍 打开或转开。 4.机床周围应避免有高频设备、电焊机等电磁干扰源，亦应避免与有 大起动电流的设备共用一套电源。 1.1.3 安装须知 为了安全，安装机床时须注意以下述事项： 1.连线 （1）必须使用本说明书中规定的套线或性能更优的套线。 （2）严禁将本机床电源与高电磁设备连接在一起。如：高频淬火机、电弧焊等。 （3）应安排有经验的合格的电气专业人员负责接线。
自动车床操作说明
宁波有限公司 一、准备工作 1、工程名称：车削。 2、使用设备：自动车床。 3、使用工具：机械配属的常用工具。 4、使用测量仪器：游标卡尺，千分尺及其它相关的测量仪器。 二、操作前注意事项： 1、依作业指导书之规定，工作前必须戴好劳动保护品、女工戴好工作帽、不准围围巾、禁止穿高跟鞋。操作时不准戴手套、不准吸烟、不准与他人闲谈、精神要集中。 2、接通电源，查看电源指示灯是否亮，并检查电路是否正常。 3、查看主轴箱内润滑油是否足量，不足时给予补充。 4、查看液压油是否足量，不足时给予补充（请用46#抗磨液压油）。 5、检查刀具是否需要研磨。 6、查看夹具是否能正常夹持。 7、给润滑部位加油润滑。
8、根据所需加料长短和大小调整送料行程和挡销高度。 9、上料。 三、开机注意事项： 1、启动油泵，给工作系统提供动力，否则无法进行运动。 2、手动调整封口长度。 3、手动启动车削电机，调整托板使工件达到要求尺寸和精度。 4、前面几项调试好以后，执行半连动加工，看车削效果是否良好，边车削边调整相 应机构，直到加工出理想工件。 5、完全调整好以后，方可选择自动进行加工。 6、加工过程中如若出现异常情况，应立刻按下急停按钮，并查找原因，排除故障以后方可继续加工。 四、停机操作： 1、加工完后取出夹具内工件，使夹头处于放松状态。 2、切断电源。 3、进行清洁保养。 五、润滑及保养： 1、机床运行中由于油温升高，可能导致油管接头渗油现象，此时应对整机油管接头重新拧紧一遍。 2、托班上的各油孔给予每班注油2次，每次在注油管有油的情况下压下手油泵2~3下。 3、油箱内油液不得低于油标视口，不足时立即给予补足，油液从上一次更换之日起每间隔半年更换一次。
U盘电路板结构图解说明及简单维修方法
U盘电路板结构图解说明及简单维修方法（缺少图） U盘的结构比较简单，主要是由USB插头、主控芯片、稳压IC(LDO)、晶振、闪存(FLASH)、PCB板、帖片电阻、电容、发光二极管(LED)等组成。 USB插头：容易出现和电路板虚焊，造成U盘无法被电脑识别，如果是电源脚虚焊，会使U盘插上电脑无任何反映。有时将U盘摇动一下电脑上又可以识别，就可以判断USB 插口接触不良。只要将其补焊即可解决问题。 稳压IC：又称LDO，其输入端5V，输出3V，有些劣质U盘的稳压IC很小，容易过热而烧毁。还有USB电源接反也会造成稳压IC烧毁。维修时可以用万用表测量其输入电压和输出电压。如无3V输出，可能就是稳压IC坏了。但有一种情况，输出电压偏低，且主控发烫，这时就是主控烧了。还有些U盘会在USB+5V和稳压IC之间串一个0欧姆的
保护电阻，此时稳压IC没有5V输入电压就是它坏了。现在许多主控都将LDO集成到主控内部了，所以我们会看到许多U盘都没有外置LDO了，它们都是USB+5V电压直接输入。这种情况就要换主控了。 晶振：早期的U盘大多都是用6M的晶振，现在的U盘则普遍采用12M晶振。晶振不耐摔，所以它是U盘上的易损件，最好的维修方法就是用相同频率的晶振直接代换。 主控芯片：主控制芯片负责闪存与USB连接，是U盘的核心，我们一般所说的U盘方案就是指主控芯片的型号。量产工具也是与它对应的。有些主控芯片还要输入3V的电压给FLASH供电，保证闪存的正常工作。 FLASH焊盘：它的作用是固定闪存，使闪存与主控连接。受外力挤压后容易使闪存与焊盘接触不良，这时会造成电脑上的U盘打不开，无法存储文件等。只要将闪存的引脚补焊一下就可以修复，也即我们常说的拖焊。 u盘结构图
机床操作说明书
机床操作说明书 5.2数控系统CNC操作面板介绍 1.TESET键：解除报警，CNC复位 2.HELP键：用于显示如何操作机床 3.DELETE键：程序编辑键 4.INPUT键：用于编辑程序和修改参数等操作 5.ALTER键：编辑程序时用 6.CAN键：删除输入到缓存的数据字母 7.INSERT键：在MDI方式操作时，输入程序 8.SH IFT键：此键用来选择键盘上的字符 9.光标键：光标的前后左右移动键 10.SYSTEM键：显示系统画面 11.POS键：显示位置画面 12.MESSAGE键：显示信息画面 13.PROG键：显示程序画面 14.CUSTOM键：显示用户宏画面 15.OFFSET SETTING键：显示刀偏/设定（SETTING）画面 详细介绍见随机提供的原版《Oi MATE Tc 操作使用说明书》 5．3 机床控制面板介绍 注意： 以下介绍各按钮的功能的前提条件是： a)机床电源总开关是在ON的状态下 b)数控系统和机床无任何报警的状态下 c)数控系统处于运行的状态下 1：系统启动，停止按钮： 按下系统启动键，10-50秒后，LCD显示初始画面，等待操作。当急停按钮按下时，LCD将显示报警。系统启动按钮主要功能是系统上电。
按下系统停止按钮，系统断电，LCD将立即无显示。关闭机床总电源时，首先关闭系统电源，然后关闭机床电源。 2：紧急停止按钮 紧急停止按钮按下时，LCD显示报警，顺时针旋转按钮释放，报警将从LCD 消失。要强调的是，当机床超过行程，压下限位开关（选项）时，在LCD上也显示报警。（装有硬限位的前提下） 3：空行程 仅对自动方式有效，机床以恒定进给速度运动而不执行程序中所指定的进给速度。该功能可用来在机床不装工件的情况下检查机床的运动。通常在编辑加工程序后，试运行程序时使用。 4：跳选 跳过任选程序段或附加任选程序段，仅对自动方式有效 5：工作方式选择 数控系统共有5种工作方式，可用工作方式选择开关或按钮选择，本机床采用触摸面板按键。 A：编辑方式 在程序保护开关通过钥匙接通的条件下，可以编辑、修改、删除或传输工件加工程序。 B：自动方式 在已事先编辑好的工件加工程序的存储器中，选择好要运行的加工程序，设置好刀具编置值。在防护门关好的前提下，按下循环启动按钮，机床就按加工程序运行。若使机床暂停，按下进给保持按钮，如有意外事件发生，按下紧急停止按钮。 C：MDI方式 MDI方式也叫手动数据输入方式，它具有从CRT/MDI操作面板输入一个程序段的指令并执行该程序段的工程。 D：JOG方式
可编程器实验板使用说明
可编程实验板使用说明 该可编程器件实验板是以Altera公司的MAX II系列可编程器件 EPM1270T144C5为核心芯片，是一款具有多种外部接口和显示器件的通用数字电路实验平台。直接连接计算机的USB接口即可对其进行编程开发并可以完成普通数字电路实验及数字可编程电路实验。 实验板长24.3CM，宽12.95CM采用FR4玻纤材料制成，环保无公害。实验板底层安装有机玻璃板方便观察实验现象并能有效避免学生在试验中因不慎将PCB板底面接触导电物体而造成的设备损坏。实验板底面的有机玻璃板还能防止学生在实验时不小心被PCB板底面的插接件针脚划伤手。 实验板采用双路电源供电方式，提供DC电源插座供电（满足7~12V直流输入）和USB-B接口电源供电。具有过流、过压和极性反转保护保护功能。 全局时钟提供9种频率可选，具体的频率值可以根据用户需求进行现场设置。输出的频率可以在板子上进行实时选择，并能通过8个LED灯的状态指示当前选择的时钟。 实验板上提供USB－Blaster下载电路。对外接口为USB-B接口。该下载电路可以直接被QuartusII软件提供的驱动程序驱动，并可以直接采用QuartusII 软件下载。提供下载所需的USB连接线。 实验板上包含16路高亮LED灯，这16路LED灯中红、黄、绿、蓝4色LED 灯每种4个交替排列，方便完成十字路口交通灯实验。 实验板包含8个7段数码管并通过扫描接口方式控制，包含1个8×8红绿双色点阵，1602字符液晶模块，包含发声器件模块，可调节声音大小，可关闭声音输出，包含8个独立按键、8个拨码开关和1个4x4矩阵键盘（扫描接口方式）。提供1个PS2 接口支持PS2键盘操作、两个独立的9针串口(DB9)、1个15针VGA接口(DB15)。 实验板包含I2C总线模块，含有I2C接口的温度传感器DS1775模块、E2PROM 模块AT24C02、实时时钟模块DS1338。 实验板提供AD模块和DA模块。AD模块采样12bit分辨率的ADS7816芯片，速度大于200KHz。DA模块采用具有2路模拟电压输出的DA芯片TLV5638，其分辨率为12bit。 可编程芯片的所有IO口均引出到接插件上，便于使用仪表进行测试。 可编程器件EPM1270没有使用的IO口，全部引出作为可扩展的外部接口使用，每组扩展接口提供电源和地接口，便于连接其他扩展模块或自搭电路。产品实物如图1-1所示：
mach3说明书
USB运动控制卡(AKZ250版本)安装手册 Ver1.17 本卡特点： ?支持Mach3 所有版本，包括目前最新版Mach3 R3.042.040。 ?支持所有Windows版本，包括目前最新版Windows7。 ?USB无需安装驱动，所有Windows版本即插即用。 ?全面支持USB热插拔，随时监测USB连线状态，Mach3工 作中，USB电缆拔出再插上，也可正常连线。 ?支持4轴联动，包括点动4轴联动。 ?支持自动对刀，电子手轮，软件限位，软件消回差功能。 ?48M速度，无需PC介入，信号由运动控制卡独立完成处理， 确保您拥有真正地实时性和可靠性。 ?拥有200KHz输出，接伺服/步进。 ?拥有状态指示灯，可提示USB连线，Mach3连线，运行中， 各类状态一目了然。 ?拥有16个输入指示灯，清楚显示信号输入状态。 ?拥有测速功能，主轴实际转速在Mach3界面中实时显示，并 且创新提供实时转速的图表显示，主轴的转速变化清晰且生动。 ?拥有板载隔离电源，无需外接电源，简化电控箱电源要求， 方便接线，同时也可使用外部电源，灵活选择。 ?采用10Mhz高速光耦10个，通用光耦24个，总计光耦达到 34个，隔离所有输入/输出，高成本设计提供完整抗干扰性能以及完善的安全保护。 ?提供完备的安装手册，文档清晰，图文并茂，描述详细。 ?电路板精心布线，唯选优质器件，制作精良。
安装手册导览 文档更新记录 运动控制卡配线示意图 外形及安装孔机械尺寸 1.安装准备 2.Mach3的软件配置 3.运动控制卡的硬件安装 https://www.wendangku.net/doc/eb5443111.html,B运动控制卡的接线表 https://www.wendangku.net/doc/eb5443111.html,B运动控制卡的接线图 6.外部倍率旋钮 7.主轴调速模拟量输出 8.主轴测速功能 9.自动对刀 10.电子手轮 11.预读缓冲设置
电路板焊接流程及其注意事项
一、电路板焊接流程及其注意事项 1、焊接微小器件（电阻、电容等）。 2、焊接电源部分，并进行电源的调试，确保各组电源的正确无误。 3、焊接IC。 4、焊接接插件。 5、电路焊接完毕，酒精浸泡10分钟左右，用刷子洗刷干净，晾干。 6、电路板的检查：A、元件有没有错焊、漏焊。 B、元件的方向、极性是否正确。 C、仔细检查是否有短路和虚焊。 注：电路板检查应重复两三次。 二、电路板焊接工艺要求： 1、正确：保证每个元件的正确无误。 2、美观：元器件摆放端正，焊接点圆滑。 3、牢固：保证元器件焊接牢固可靠。 三、整机测试 1、编码器的测试： 准备工作：准备好调制器一台，节目源（如DVD等）一台，卫星接收机（用作接收调制器信号）一台，电视机一台，视频线三根，S端子连接线、音频线、码硫数据线（双Q9线）、射频线（L16转F 头）各一根。
测试步骤： A.连接好各种数据线和电源线，开机，查看显示屏，看显示是否正常。 B.操作键盘，首先将编码器调用一次默认设置。 C.在电视机上查看图像效果，看图像是否正常。 D.用电吹风给编码器慢慢加温，观察图像是否正常。 E.用手敲打编码器，观察图像是否正常。 F.重复多次开关机，看编码器是否很正常工作。 G.接口检查：更换不同的数据接口进行检查。 注：以上操作过程所涉及的具体操作方法请查看产品操作说明书，对应说明书所注明的功能做一次检查。 测试结束：在以上检查过程，图像和声音一直是流畅的设备为合格设备。 2、复用器测试： 准备工作：准备好调制器一台，编码器一台，节目源（如DVD 等）一台，卫星接收机（用作接收调制器信号）一台，电视机一台，码流数据线两根，视频线、音频线、（双Q9线）、射频线（L16转F 头）各一根。
http接口说明模板
一、查询菜品列表接口(DONE) 1、功能说明 接受提供的菜品 XXX画面XXX功能（比如人口查询画面-查询| 人口查询画面-详细等）2、接口调用说明 获取地址 请求方式 GET 数据返回格式 JSON 传递参数
成功返回结果 [ “serverResponse”:”Success”, “totalRecords”:”52”, “page” : “10”, “pageSize” : “5”, “data”：{ “id” : “123”, “itemName” : “皇堡”, “priceNow” : “10”, “pricePast” : “12”, “servicePicture” :””, “serviceStars” : “3” }, ……. { “id” : “”, “itemName” : “”, “priceNow” : “”, “pricePast” : “”, “servicePicture” :” ”, “serviceStars” : “3” } ]
返回结果解释 二、查询广告接口（DONE） 1、功能说明 接受广告图片，目前为一张 2、接口调用说明 获取地址 请求方式 GET 数据返回格式 JSON
传递参数 成功返回结果 { "serverResponse":"Success", “advertisePicture” : “” } 返回结果解释 三、查询订单列表接口(DONE) 1、功能说明 根据指定的客户id查询订单
2、接口调用说明 获取地址 请求方式 GET 数据返回格式 JSON 传递参数 参数说明 这个接口可以作为多种用途： 搜索正在进行中的订单：customerID和orderStatus 成功返回结果 { "serverResponse":"Success", “data”：[ { “orderNo” : “02135” “orderTime” : “11:00”,
CNC-机床说明书及维护手册讲课讲稿
6-2 操作面板功能說明 ◆本節說明機械操作面板上各按鍵與開關之功能，按鍵與開關之位置如圖所示： 模式模式說明功能說明圖例備註 DNC 個人電腦聯線 模式 機台可以一方面與個人電腦執 行程式傳輸，另一方面可以同 時加工 按CYCLESTART啟動 按FEEDHOLD暫停 EDIT 內部記憶體 程式編輯模式 可以編輯新的加工程式，也可 以修改記憶體中舊的加工程 式.(三菱系統在所有模式下都 可以編輯修改) AUTO 記憶體模式機台可以執行體中的加工程 式.(應先在MONITOR書面下將 程式號碼呼叫了來) 按CYCLESTART啟動 按FEEDHOLD暫停 MDI 手動操作模式可在MDI書面下,輸入簡易加 工程式,並加以執行 按CYCLESTART啟動 按FEEDHOLD暫停 HANDLE 手輪操作模式機台各軸的移動可借手輪操作盒上的軸選擇開關及倍率加以控制 JOG 寸動模式機台各軸的寸動移動,可籍軸選擇開關、移動方向選擇開關及移動速率選擇開關加以控制 RAPID 快速移動模式機台執行各軸的快速移動,可 借軸選擇開關、移動方向選擇 開關及快速移動速率選擇開關 加以控制. 各軸原點複歸完成 前,請勿以50%或100% 快速移動速率操作 ZRN 原點複歸模式機台各軸執行原點複歸功 能,(可選擇Z軸先原點複歸 後,其他軸才能執行原點複歸 功能) 務必考慮各軸的原點複 歸順序,避免撞機危險
◆軸的移動方向移動速率選擇 開關名稱功能說明圖例有效模式 JOG/FEEDRATE OVERRIDE 各軸的寸動及切削移動速率選 擇開關,JOG模式下各軸的移動 速率 mm/min為單位, DNC/AUTOMDI 等模式以%為單位 JOG/DNC/ AUTO/MDI RAPID OVERRIDE 快速移動速率選擇開關 RAPID/ZRN/DNC/ AUTO/MDI +X +X 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN -X -X 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN +Y +Y 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN -Y -Y 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN ◆軸的移動方向移動速率選擇 開關名稱功能說明圖例有效模式+Z +Z 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN -Z -Z 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN +4 +4 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN -4 -4 軸的移動方向選擇按鈕開關JOG/RAPID/ZRN ◆主軸控制功能 開關名稱功能說明圖例有效模式SPENDLE OVERRIDE 主軸旋轉轉速調整DNC/AUTO/MDT SPINDLE CW 主軸CW旋轉HANDLE/JOG/RAPID/ZRN SPINDLE STOP 主軸停止旋轉HANDLE/JOG/RAPID/ZRN SPINDLE CCW 主軸CCW旋轉HANDLE/JOG/RAPID/ZRN SPINDLE ORT 主軸定位HANDLE/JOG/RAPID/ZRN