肠杆菌科AcrAB多药外排泵分子演化分析

目的：分析肠杆菌科AcrAB多药外排泵的分子演化规律,为多药耐药性病原菌的防治提供基础数据。方法：从NCBI获得肠杆菌科物种AcrAB多药外排泵相关蛋白和核酸序列,采用分析软件,分析肠杆菌科物种AcrAB多药外排泵相关序列。结果：在肠杆菌科各物种AcrA、AcrB和AcrR与大肠埃希氏菌同源性在55%、75%和43%以上。AcrA保守位点分散,在N端和C端较少,在分子一级结构中段较多。AcrB跨膜序列保守性较高,与质子转移相关的三个位点D407、D408和K940以及稳定这三者结构的T978在肠杆菌科完全保守,其一级结构上相邻位点也保守。AcrR序列整体保守性较低,但HTH区域保守性高。与AcrR结合的回文结构及周围序列保守性高,在＂茎＂结构中仅存在一个氨基酸的差异。结论：AcrAB多药外排泵在肠杆菌科中广泛存在,有一定的保守性。分析肠杆菌科AcrAB多药外排泵有助于病原菌多药耐药性的防治。     

氨基酸残基可及性与蛋白质家族成员结构的保守性

本文在细胞色素ｃ族蛋白和免疫球蛋白家族中一些蛋白质片段的序列比较和分析的基础上,通过计算其氨基酸残基的可及性,对残基可及性与蛋白质序列及其三维结构的保守性之间的关系进行了分析和探讨。结果表明,序列中凡是保守的残基,其可及性都较低,而且这些低可及性的保守性残基与维持蛋白质特有的三维结构相关。作者认为,同一家族的蛋白质中,在进化上相距较远的各成员之间,结构的保守性主要是体现在其三维结构上;序列中的保守     

植物Ⅲ型聚酮合酶基因家族的分子进化分析

Ⅲ型聚酮合酶(type Ⅲ polyketide synthase,PKSⅢ)广泛存在于细菌、真菌和植物中,目前数据库中已积累了大量的序列资料。为了进一步了解植物Ⅲ型聚酮合酶基因家族的分子进化,以及其作为系统进化研究材料的可能性,选取了75条来自不同植物物种包括苔藓类植物、蕨类植物、裸子植物、单子叶植物和双子叶植物的PKSⅢ蛋白序列,用CLUSTAL X软件对其氨基酸序列进行了比对,并用邻位相接法构建了系统进化树。结果表明,尽管不同来源的PKSⅢ序列表现了很大的差异,但保守结构域CHS-like所包含的主要功能位点半胱氨酸(Cys184)、苯丙氨酸残基(Phe236和Phe286)、组氨酸残基(His335)、天冬酰氨残基(Asn369)在各植物物种中具有很好的保守性;同时发现,在植物PKSⅢ序列中多数的Cys位点均具有较好的保守性,而且蕨类植物PKSⅢ和单子叶植物PKSⅢ在Cys保守位点有很好的相似性;进一步构建分子进化树表明,PKSⅢ基因基本上首先根据功能而聚类,明显地划分为CHSs和non-CHSs两类,其次按照不同的植物物种聚类。     

草鱼RBM cDNA克隆及序列分析

目的:克隆草鱼RNA剪切蛋白(RNA binding motif protein-RBM)基因.方法:采用RT -PCR技术从草鱼性腺总RNA中克隆RBM基因,将其插入pBS-T载体上,经菌液PCR鉴定后进行 测序并对测序结果进行生物信息分析.结果:获得草鱼RBM cDNA部分序列 ,大小为384bp,预 测编码127个氨基酸残基;RBM基因进化与物种形态进化类似;不同物种间RBM基因一级结构 保守性不高,但氨基酸序列保守性较高,且存在高度保守的氨基酸位点.结论: 成功地克隆了草鱼RBM cDNA部分序列,为获RBM cDNA全长序列及后续研究提供理论基础.     

氨基酸残基归类及用简化后的字符识别蛋白质结构保守区域

序列比对是寻找蛋白质结构保守性区域的常用方法, 然而当序列相似小于30%时比对准确度却不高, 这是因为在这些序列中具有相似结构功能的不同残基在序列比对中往往被错误配对. 基于相似的物理化学性质, 某些残基可以被归类为一组, 而应用这些简化后的残基字符可以有效地简化蛋白质序列的复杂性并保持序列的主要信息. 因此, 如果20种天然氨基酸残基能够正确的归类, 可以有效地提高序列比对的准确度. 本文基于蛋白质结构比对数据库DAPS, 提出了一种新的氨基酸残基归类方法, 并可以同时得到不同简化程度下的替代矩阵用于序列比对. 归类的合理性由相互熵方法确认, 并且应用简化后的字符表于序列比对来识别蛋白质的结构保守区域. 结果表明, 当氨基酸残基字符简化到9个左右时能够有效地提高序列比对的准确度.     

甜荞FaesSTK基因在长花柱长雄蕊突变体lpls中的表达分析

采用RACE技术,从甜荞（Fagopyrum esculentum Moench）中克隆获得3种花型的STK同源基因FaesSTK,并对其序列特征进行分析。结果显示,甜荞3种花型植株STK同源基因序列一致,全长为967 bp,包含长689 bp的完整开放阅读框,编码一个由225个氨基酸残基组成的D类MADS-box转录因子。蛋白序列比对及系统发育分析结果表明,FaesSTK蛋白属于MADS-box转录因子中的STK进化系。包含1个由57个氨基酸残基组成的高度保守的MADS结构域;1个由82个氨基酸残基组成的次级保守区域的K结构域,在C端的转录激活区还含有另外2个高度保守的基序（AGⅠ和AGⅡ）。实时荧光定量检测结果显示,FaesSTK基因主要在甜荞lpls突变体的雄蕊、雌蕊和不同发育时期的幼果中表达,在根和花被片中仅能检测到微弱的转录信号,在叶和茎中不表达,其中在雌蕊和果实中的表达量极显著高于其他组织。推测该基因在花发育过程中可能主要参与调控甜荞lpls突变体雌蕊和果实的发育。     

甜荞FaesSTK基因在长花柱长雄蕊突变体lpls中的表达分析

采用RACE技术,从甜荞(Fagopyrum esculentum Moench)中克隆获得3种花型的STK同源基因Faes STK,并对其序列特征进行分析。结果显示,甜荞3种花型植株STK同源基因序列一致,全长为967 bp,包含长689 bp的完整开放阅读框,编码一个由225个氨基酸残基组成的D类MADS-box转录因子。蛋白序列比对及系统发育分析结果表明,Faes STK蛋白属于MADS-box转录因子中的STK进化系。包含1个由57个氨基酸残基组成的高度保守的MADS结构域; 1个由82个氨基酸残基组成的次级保守区域的K结构域,在C端的转录激活区还含有另外2个高度保守的基序(AGⅠ和AGⅡ)。实时荧光定量检测结果显示,Faes STK基因主要在甜荞lpls突变体的雄蕊、雌蕊和不同发育时期的幼果中表达,在根和花被片中仅能检测到微弱的转录信号,在叶和茎中不表达,其中在雌蕊和果实中的表达量极显著高于其他组织。推测该基因在花发育过程中可能主要参与调控甜荞lpls突变体雌蕊和果实的发育。     

草莓光信号转导因子FaHY5的克隆及其表达分析

以‘丰香’草莓为试材,通过同源克隆法获得FaHY5 cDNA全长序列,运用生物学软件对该序列及其编码的氨基酸序列进行相关生物信息学及结构功能分析,同时采用半定量及荧光定量方法对FaHY5基因在不同组织和果实不同发育阶段的表达模式进行研究。结果表明,FaHY5全长为501 bp,Gen Bank登录号为KP984791,编码166个氨基酸。蛋白质分子量约为18.136 kD,理论等电点为10.05,C末端存在典型的bZIP保守结构域,是一个非分泌型核定位蛋白。序列比对及进化树分析,HY5在进化的过程中具有高度的保守性。表达谱分析,FaHY5在各组织中均有表达,且在花中表达量最高;而在果实发育过程中,FaHY5主要在前期积累。本研究分离鉴定了草莓中HY5(LONG HYPOCOTYL 5)转录因子并探索了其在草莓中的表达模式,为进一步研究草莓FaHY5转录因子的功能和作用机制提供理论基础。     

细胞色素c蛋白序列分析与结构比较

首先以马心细胞色素c(Horse Cytc)蛋白的氨基酸序列为查询序列,利用生物信息学方法进行相似性搜索,获得了一系列细胞色素c(Cytc)蛋白的氨基酸序列,然后对Cytc蛋白进行了多重对齐分析、进化分析和三维结构比较分析。分析结果表明:Cytc中某些特定部位的氨基酸残基高度保守;相近物种来源的Cytc具有较近的亲缘关系,而来源于同一物种不同部位的Cytc却具有较远的亲缘关系;来源于不同物种的Cytc,即使具有较远的亲缘关系,却具有极其相似的三维空间结构。这些研究结果将为基于Cytc进行蛋白分子设计与构建提供指导意义。     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Interaction between the α-barrel tip of Vibrio vulnificus TolC homologs and AcrA implies the adapter bridging model

The AcrAB-TolC multidrug efflux pump confers resistance to Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor that participates in the formation of type I secretion systems. The genome of Vibrio vulnificus encodes two proteins homologous to the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complement the E. coli TolC function and physically interact with the membrane fusion protein AcrA, a component of the E. coli AcrAB-TolC efflux pump. Using site-directed mutational analyses and an in vivo cross-linking assay, we demonstrated that the α-barrel tip region of TolC homologs plays a critical role in the formation of functional AcrAB-TolC efflux pumps. Our findings suggest the adapter bridging model as a general assembly mechanism for tripartite drug efflux pumps in Gram-negative bacteria.     

Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system

The major Escherichia coli multidrug efflux pump AcrAB-TolC expels a wide range of antibacterial agents. Using in vivo cross-linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate-induced assembly of the closely related TolC-dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross-linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo. Confirming this view, AcrA could be cross-linked independently to AcrB and TolC in vivo, and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C-terminal half of AcrA, but binding to TolC required interaction of N- and C-terminal polypeptides spanning the lipoyl-like domains predicted to present the intervening coiled-coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug-independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.     

Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump

AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that, among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues 357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at, its C terminus is involved in the interaction with its cognate pump protein, AcrB.     

Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.     

A model of a transmembrane drug-efflux pump from Gram-negative bacteria

In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric inner-membrane proton-antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.     

Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and β-barrel domains of AcrA.     

Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.     

AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex

Many transporters of Gram-negative bacteria involved in the extracellular secretion of proteins and the efflux of toxic molecules operate by forming intermembrane complexes. These complexes are proposed to span both inner and outer membranes and create a bridge across the periplasm. In this study, we analyzed interactions between the inner and outer membrane components of the tri-partite multidrug efflux pump AcrAB-TolC from Escherichia coli. We found that, once assembled, the intermembrane AcrAB-TolC complex is stable during the separation of the inner and outer membranes and subsequent purification. All three components of the complex co-purify when the affinity tag is attached to either of the proteins suggesting bi-partite interactions between AcrA, AcrB, and TolC. We show that antibiotics, the substrates of AcrAB-TolC, stabilize interactions within the complex. However, the formation of the AcrAB-TolC complex does not require an input of energy.     

The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC

Periplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain of Escherichia coli AcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.AcrA, the multidrug efflux protein from Escherichia coli, is the best-characterized member of the membrane fusion protein (MFP) family (24). Periplasmic AcrA associates with the inner-membrane transporter AcrB, belonging to the RND superfamily of proteins, and the outer-membrane factor TolC (22, 23). Together, the three components form a transenvelope multidrug efflux pump responsible for the high levels of intrinsic as well as acquired antibiotic resistance of E. coli.AcrA is anchored into the inner membrane by N-terminal lipid modification. However, genetic complementation studies showed that the presence of the lipid moiety is not required for AcrA function (14, 24). Structural studies of the proteolytically stable core of AcrA (amino acid [aa] residues 46 to 312) and of whole-length MexA, a homologous protein from Pseudomonas aeruginosa, showed that these proteins have modular structures (Fig. ​(Fig.1A).1A). They comprise the α-helical hairpin, the lipoyl-binding domain, and the α-β-barrel domain (2, 9, 14). Mutagenesis and chemical cross-linking studies identified the α-helical hairpin of AcrA as a TolC-binding domain, whereas the α-β-barrel domain was proposed to bind AcrB (6, 11, 12, 16). Surprisingly, in isothermal calorimetry experiments, the core fragment of AcrA without its C-terminal domain (C-domain) was able to bind neither AcrB nor TolC (23). In contrast, the whole-length AcrA interacted with both components. This result suggested that the C-domain of AcrA might be important for these interactions. In crystal structures, however, the C-domains of AcrA and MexA were not resolved, and their structures remain unknown.Open in a separate windowFIG. 1.Proteolytic profiles of AcrA^his in vitro and in vivo. (A) Schematic representation of the secondary structure of AcrA. The unique N-terminal Cys25, which is lipid modified after processing in the periplasm, is shown with an arrow. Positions of amino acid residues that form the α-β-barrel, lipoyl-binding, and α-helical hairpin domains are indicated. AcrA residues cleaved by trypsin are indicated by arrowheads. The 28.9-kDa (K46-R315) core and the 26.5-kDa fragment (K46-R294) are also indicated. (B) Purified AcrA^his (final concentration, 1.95 μM) was digested with trypsin (final concentration, 0.10 μM) at 37°C. Aliquots (10 μl) were taken at different time points, and reactions were terminated by boiling in the SDS sample buffer for 5 min. Tryptic fragments were resolved by SDS-PAGE and analyzed by silver nitrate staining. Minor fragments in the untreated control (0 min) are contaminants that copurify with AcrA^his. Lane M, molecular marker. (C) Proteolytic profiles of AcrA^his in E. coli AG100AX cells carrying pA^his and pA^hisB plasmids. After treatment with increasing concentrations of trypsin for 60 min at 37°C, the whole-cell proteins were resolved by SDS-PAGE and analyzed by immunoblotting with a polyclonal anti-AcrA antibody. Masses of tryptic fragments of the C-domain of AcrA^his identified by mass spectrometry and by mobility in SDS-PAGE are indicated. O.D., optical density as determined by absorbance at 600 nm.The alignment of sequences of highly diverse MFPs from both gram-negative and gram-positive bacteria showed that amino acid sequences of the C-domains are conserved among members of the MFP family (4). In addition, several studies suggested that this region is important for the function of AcrA. The deletion mutant of AcrA lacking 85 C-terminal aa residues is poorly expressed and nonfunctional in multidrug efflux (14). The replacement of aa 290 to 357 of AcrA with an analogous region of YhiU disrupted AcrA function possibly because of the loss of interaction with the AcrB transporter (5). Random mutagenesis of MexA identified C-terminal amino acid residues as important for MexA oligomerization and interaction with MexB (16, 17).In this study, we identified proteolytically labile sites in the C-domain (aa 315 to 397) of the purified AcrA and compared the accessibility of these sites to that in free AcrA and when engaged in the bipartite and tripartite AcrA, AcrB, and TolC interactions in vivo. We found that the assembly of the AcrAB-TolC complex, but not bipartite AcrA-AcrB and AcrA-TolC interactions, protects the C-domain of AcrA from proteolytic digestion. This result suggested that this domain of AcrA interacts with AcrB, TolC, or both. The functional significance of the C-domain was confirmed by site-directed mutagenesis. A single G363C substitution significantly impairs the multidrug efflux activity of AcrAB-TolC.     

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.