Changing the Transport of a Cell

Abstract

The review deals with some of the transport functions of different systems that have been implicated with several pathological disorders. Membrane transport role in parasitic diseases and metal resistance is discussed as a few selected examples. Among various limitations that are encountered in recombinant technology and in heterologous expression of proteins, transport functions of the host organisms cannot be ignored. Recently, membrane transport has acquired a new emerging role in multidrug resistance. Several membrane transporters, particularly ATP binding cassette (ABC) proteins that are involved in drug resistance, have been identified throughout the evolutionary scale. The review briefly emphasizes that membranes are not only important as structural elements but are also adopted to perform diverse functions.     

The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation

Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer.     

Nucleotide Sequence and Genetic Characterization of the Novel IncQ-like Plasmid pIE1107

The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids. Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems. However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons. Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons. Experiments with clonedoriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its ownoriV which differs only by a few nucleotides from theoriV of IncQ plasmids. Implications from the apparent highly specific protein–DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed.     

Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.     

Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics and Potential Applications in Biotechnology

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.     

Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.     

Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture

The arabinose-inducible promoter P(BAD) is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible P(tac) and P(taclac) promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P(BAD)) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.     

Identification of the btuCED polypeptides and evidence for their role in vitamin B12 transport in Escherichia coli.

Passage of vitamin B12 across the outer and cytoplasmic membranes of Escherichia coli occurs in two steps, each involving independent transport systems. Since the vitamin accumulated in btuC or btuD mutants is readily released from the cell by chase or osmotic shock and does not undergo the usual metabolic conversions, the products of these genes might participate in transport across the cytoplasmic membrane. Mutations in btuC and btuD are complemented by recombinant plasmids carrying a 3,410-base-pair HindIII-HincII DNA fragment. Transposon Tn1000 mutagenesis and subcloning defined the location of these two genes and showed that they are separated by approximately 800 base pairs. The polypeptides elicited by this fragment and its derivatives were identified by using a maxicell system. The apparent molecular weight of the btuC product was approximately 26,000, that of the btuD product was 29,000. Both polypeptides were associated with the cell membrane. Transposon insertions in the region between btuC and btuD, as well as those in the two genes, conferred a deficiency in vitamin B12 utilization and transport when they were crossed onto the chromosome. This region, termed btuE, encoded a 22,000-Mr polypeptide and lesser amounts of a 20,000-Mr species. A portion of the BtuE protein was released from maxicells by osmotic shock or spheroplast formation. The relative production of BtuE and BtuD in response to plasmids carrying transposon insertions suggested that the three genes are arranged in an operon in the order btuC-btuE-btuD and that internal promoters exist since polarity was incomplete. Substantial elevation of transport activity was engendered by plasmids carrying the intact btu region, but not when any of the btu genes was disrupted. The btuCED region thus may encode a transport system for passage of vitamin B12 across the cytoplasmic membrane. This system bears similarities to periplasmic binding protein-dependent transport systems, although the putative periplasmic component is not required for its function.     

毒性分子-抗毒性分子系统的生物学作用研究进展

毒性分子-抗毒性分子系统(toxin-antitoxin systems,TA systems)被发现广泛存在于细菌染色体、质粒以及古细菌基因组中。TA系统是由2个基因组成的操纵子,这2个基因分别编码稳定的毒性分子和不稳定的抗毒性分子。毒性分子总是蛋白质,抗毒性分子可能是蛋白质或RNA。因此,根据抗毒性分子的性质和作用方式的不同可将TA系统家族分为5种类型。Ⅰ型和Ⅲ型的抗毒性分子是RNA,能抑制毒性分子的合成或者与其隔离;II、IV和V型的抗毒性分子是蛋白质,能隔离、平衡毒性分子作用或抑制其合成。TA系统具有多种生物学功能。目前研究表明,TA系统可能在细菌应激应答、程序化细胞死亡、多重耐药的形成、防止DNA入侵、稳定大基因组片段等方面有重要的作用。     

Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens.

The occ and noc regions of octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of octopine and nopaline, respectively. Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regions contained additional DNA stretches which were under the control of opine-inducible promoters, but the functions were unknown. We investigated these stretches by DNA sequence and functional analyses. The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction. The occ and noc region genes are related, but the arrangement of the genes is different. The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria. The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein. We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled octopine showed that the putative transport genes in the occ region code for octopine uptake proteins. The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region.     

Hydroxamate-mediated transport of iron controlled by ColV plasmids.

A new high-affinity system for iron transport, associated with the presence of ColV plasmids, has been detected in Escherichia coli and partially characterized. The presence of such "iron-transport plasmids" in E. coli cells that are defective in enterochelin-mediated transport of iron enabled them to grow in media to which 2,2'-dipyridyl had been added to reduce availability of iron. In addition, the presence of plasmid deoxyribonucleic acid in a mutant defective in enterochelin biosynthesis was associated with a marked increase in the rate of radioactive-iron uptake. Plasmid-determined uptake of iron was distinct from previously recognized systems for iron transport in E. coli K-12, and the colicin V molecule appeared not to be directly involved. Hydroxylamine-nitrogen could be detected in cell pellets of ColV+ cultures, and similar material was detected in supernatant fluids of late log- or stationary-phase cultures. The hydroxamate material was not detected in cell pellets or culture supernatants of strains from which plasmids had been eliminated, and a 95% decrease in hydroxamate synthesis was observed when cells were grown in minimal medium containing 2 microM iron.     

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.     

Photosensitization of wild and mutant strains of Escherichia coli by meso-tetra (N-methyl-4-pyridyl)porphine

Wild type Escherichia coli cells as well as some mutant strains lacking specific DNA repair systems are efficiently killed upon visible light-irradiation after 5 min-incubation with meso-tetra(4N-methyl-pyridyl)porphine (T4MPyP). The presence of oxygen is necessary for cell photoinactivation. The porphyrin appears to exert its phototoxic activity largely by impairing some enzymic and transport functions at the level of both the outer and cytoplasmic membrane. Thus, SDS-PAGE electrophoresis shows a gradual attenuation of some transport protein bands as the irradiation proceeds, while a complete loss of lactate and NADH dehydrogenase activities is caused by 15 min-exposure to light. On the other hand, DNA does not represent a critical target of T4MPyP photosensitization as suggested by the closely similar photosensitivity of the wild E. coli and E. coli strains defective for two different DNA repair mechanisms, as well as by the lack of any detectable alteration of the pUC19 plasmids extracted from photosensitized E. coli TG1 cells.     

DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.     

整合子-基因盒系统与细菌耐药机制的研究进展

大量研究表明整合子-基因盒系统是微生物耐药的主要机制,由其介导的耐药基因水平转移是细菌耐药机制产生的主要途径。已知的整合子被分为两大类：传统的整合子和超级整合子。前者存在于转座子、质粒和细菌染色体,其基因盒编码产物可使细菌耐受一种或多种抗菌药物及消毒剂;而后者则只存在于细菌的染色体上,它携带的基因盒更多,且其编码产物则更加复杂,目前只在特定菌株中发现超级整合子。本文就整合子的结构、分布、检测及它对细菌耐药性的影响等几个方面的研究进展进行讨论。     

Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC’s survival in the host’s low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.     

Periplasmic binding protein-dependent transport systems: the membrane-associated components

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.     

Arsenical resistance in the IncHI2 plasmids

The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100mM) and sodium arsenite (up to 10mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis.     

Bacterial resistance and detoxification of heavy metals

Microbial cells have resistances to essentially all of the toxic heavy metals of the Periodic Table. In bacterial cells, the genetic determinants of these resistances are frequently found on small extrachromosomal plasmids and transposons. Sometimes the resistances are associated with detoxifying enzymes. This is true for the Hg²⁺ → Hg⁰ reductase, the As³⁺ → As⁵⁺ oxidase and the Cr⁶⁺ → Cr³⁺ reductase. In other cases, such as As⁵⁺, Ag⁺ and Cd²⁺, no change in redox state occurs but, rather, uptake and transport differences accompany resistance determinants. This article summarizes what is known of bacterial metal resistances for which enzymatic detoxification is known to be the mechanism of resistance. The characteristics and functions of the enzymes are described, as well as a summary of the newer DNA sequence analysis (basic science) and bench-scale efforts (applied science) for the mercuric resistance system.     

Cysteine metabolism in an isolate of Lactobacillus sake: Plasmid composition and cysteine transport

Abstract Lactobacillus sake L13, an isolate from vacuum packaged beef is able to utilise cysteine as an energy source for growth in rich medium. This isolate segregates into two strains, one of which retains the ability to grow on cysteine (notated as strong cells) while the other grows only marginally (weak). Both strains possess cysteine desulfhydrase activity, but only one is able to actively transport cysteine (high affinity). Uptake is sensitive to inhibition by valinomycin, CCCP, nigericin and arsenate. Comparison of the two cell types indicates that there are two cysteine transport systems and that the alternative one is passive and of low affinity. Strong cells possess two plasmids (2.7 and 8.3 kb) while the weak strain has lost the plasmids. Curing of the strong cells results in plasmid loss and loss of the high affinity cysteine uptake system.