Addressing the New Global Threat of Xylella fastidiosa.

Xylella fastidiosa is one of the most important threats to plant health worldwide. This bacterial pathogen has a long history, causing disease in the Americas on a range of agricultural crops and trees, with severe economic repercussions particularly on grapevine and citrus. In Europe, X. fastidiosa was detected for the first time in 2013 in association with a severe disease affecting olive trees in southern Italy. Subsequent mandatory surveys throughout Europe led to discoveries in France and Spain in various host species and environments. Detection of additional introductions of X. fastidiosa continue to be reported from Europe, for example from northern Italy in late 2018. These events are leading to a sea change in research, monitoring and management efforts as exemplified by the articles in this Focus Issue . X. fastidiosa is part of complex pathosystems together with hosts and vectors. Although certain X. fastidiosa subspecies and environments have been well studied, particularly those that pertain to established disease in North and South America, this represents only a fraction of the existing genetic, epidemiological, and ecological diversity. This Focus Issue highlights some of the key challenges that must be overcome to address this new global threat, recent advances in understanding the pathosystem, and steps toward improved disease control. It brings together the broad research themes needed to address the global threat of X. fastidiosa, encompassing topics from host susceptibility and resistance, genome sequencing, detection methods, transmission by vectors, epidemiological drivers, chemical and biological control, to public databases and social sciences. Open communication and collaboration among scientists, stakeholders, and the general public from different parts of the world will pave the path to novel ideas to understand and combat this pathogen.

Efficient enrichment cloning of TAL effector genes from Xanthomonas.

Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars of Xanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL effectors in their genomes. Some of the TAL effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL effector genes remains a challenge. In order to isolate complete TAL effector gene repertoires, we developed an enrichment cloning strategy based on •genome-informed in silico optimization of restriction digestions,•selective restriction digestion of genomic DNA, and•size fractionation of DNA fragments. Our rapid, cheap and powerful method allows efficient cloning of TAL effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains of Xanthomonas oryzae from Africa.

Transcriptome and proteome analysis reveal new insight into proximal and distal responses of wheat to foliar infection by Xanthomonas translucens.

The molecular details of local plant response against Xanthomonas translucens infection is largely unknown. Moreover, there is no knowledge about effects of the pathogen on the root's transcriptome and proteome. Therefore, we investigated the global gene and protein expression changes both in leaves and roots of wheat (Triticum aestivum) 24 h post leaf infection of X. translucens. This simultaneous analysis allowed us to obtain insight into possible metabolic rearrangements in above- and belowground tissues and to identify common responses as well as specific alterations. At the site of infection, we observed the implication of various components of the recognition, signaling, and amplification mechanisms in plant response to the pathogen. Moreover, data indicate a massive down-regulation of photosynthesis and confirm the chloroplast as crucial signaling hub during pathogen attack. Notably, roots responded as well to foliar attack and their response significantly differed from that locally triggered in infected leaves. Data indicate that roots as a site of energy production and synthesis of various secondary metabolites may actively influence the composition and colonisation level of root-associated microbes. Finally, our results emphasize the accumulation of jasmonic acid, pipecolic acid and/or the downstream mediator of hydrogen peroxide as long distal signals from infected leaves to roots.

High-Quality Draft Genome Sequences of Two Xanthomonas Pathotype Strains Infecting Aroid Plants.

We present here the draft genome sequences of bacterial pathogens of the Araceae family, Xanthomonas axonopodis pv. dieffenbachiae LMG 695 and Xanthomonas campestris pv. syngonii LMG 9055, differing in host range. A comparison between genome sequences will help understand the mechanisms involved in tissue specificity and adaptation to host plants.

New multilocus variable-number tandem-repeat analysis tool for surveillance and local epidemiology of bacterial leaf blight and bacterial leaf streak of rice caused by Xanthomonas oryzae.

Multilocus variable-number tandem-repeat analysis (MLVA) is efficient for routine typing and for investigating the genetic structures of natural microbial populations. Two distinct pathovars of Xanthomonas oryzae can cause significant crop losses in tropical and temperate rice-growing countries. Bacterial leaf streak is caused by X. oryzae pv. oryzicola, and bacterial leaf blight is caused by X. oryzae pv. oryzae. For the latter, two genetic lineages have been described in the literature. We developed a universal MLVA typing tool both for the identification of the three X. oryzae genetic lineages and for epidemiological analyses. Sixteen candidate variable-number tandem-repeat (VNTR) loci were selected according to their presence and polymorphism in 10 draft or complete genome sequences of the three X. oryzae lineages and by VNTR sequencing of a subset of loci of interest in 20 strains per lineage. The MLVA-16 scheme was then applied to 338 strains of X. oryzae representing different pathovars and geographical locations. Linkage disequilibrium between MLVA loci was calculated by index association on different scales, and the 16 loci showed linear Mantel correlation with MLSA data on 56 X. oryzae strains, suggesting that they provide a good phylogenetic signal. Furthermore, analyses of sets of strains for different lineages indicated the possibility of using the scheme for deeper epidemiological investigation on small spatial scales.

Draft Genome Sequence of the Flagellated Xanthomonas fuscans subsp. fuscans Strain CFBP 4884.

We report the draft genome sequence of the flagellated strain CFBP 4884 of Xanthomonas fuscans subsp. fuscans, which was isolated in an outbreak of common bacterial blight of beans along with non-flagellated strains. Comparative genomics will allow one to decipher the genomic diversity of strains cohabiting in epidemics.

Draft Genome Sequence of Xanthomonas axonopodis pv. allii Strain CFBP 6369.

We report here the draft genome sequence of Xanthomonas axonopodis pv. allii strain CFBP 6369, the causal agent of bacterial blight of onion. The draft genome has a size of 5,425,942 bp and a G+C content of 64.4%.

Analysis of Xanthomonas oryzae pv. oryzicola population in Mali and Burkina Faso reveals a high level of genetic and pathogenic diversity.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola was first reported in Africa in the 1980s. Recently, a substantial reemergence of this disease was observed in West Africa. Samples were collected at various sites in five and three different rice-growing regions of Burkina Faso and Mali, respectively. Sixty-seven X. oryzae pv. oryzicola strains were isolated from cultivated and wild rice varieties and from weeds showing BLS symptoms. X. oryzae pv. oryzicola strains were evaluated for virulence on rice and showed high variation in lesion length on a susceptible cultivar. X. oryzae pv. oryzicola strains were further characterized by multilocus sequence analysis (MLSA) using six housekeeping genes. Inferred dendrograms clearly indicated different groups among X. oryzae pv. oryzicola strains. Restriction fragment length polymorphism analysis using the transcriptional activator like effector avrXa7 as probe resulted in the identification of 18 haplotypes. Polymerase chain reaction-based analyses of two conserved type III effector (T3E) genes (xopAJ and xopW) differentiated the strains into distinct groups, with xopAJ not detected in most African X. oryzae pv. oryzicola strains. XopAJ functionality was confirmed by leaf infiltration on 'Kitaake' rice Rxo1 lines. Sequence analysis of xopW revealed four groups among X. oryzae pv. oryzicola strains. Distribution of 43 T3E genes shows variation in a subset of X. oryzae pv. oryzicola strains. Together, our results show that African X. oryzae pv. oryzicola strains are diverse and rapidly evolving, with a group endemic to Africa and another one that may have evolved from an Asian strain.

First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Uganda.

In June 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the booting stage in the Doho rice irrigation scheme, Butaleja district, and at the tillering stage in Nambale, Iganga district and Magada, Namutumba district of Uganda. In areas surveyed, disease incidence was about 80, 40, and 30% in Doho, Nambale, and Magada, respectively. Outside the irrigation schemes, it was lower but widespread. Affected leaves showed typical BLS symptoms, such as water-soaked lesions, translucent stripes, and yellow-brown to black streaks, sometimes with visible exudates at the leaf surfaces. To check for the presence of the bacteria, symptomatic leaves were ground in sterile water and the suspension obtained was subjected to a multiplex PCR assay for X. oryzae pathovars, leading to the three diagnostic DNA fragments for X. oryzae pv. oryzicola (3). In parallel, bacterial strains were isolated from surface-sterilized symptomatic leaves. To this end, rice leaves were ground in sterile distilled water and serial dilutions of the cell suspensions were plated on semi-selective PSA medium (4). Each of the three samples yielded yellow, mucoid Xanthomonas-like colonies that resembled the positive control strain MAI10 (1). These isolates were named Ug_1, Ug_10, and Ug_14, which originated from Doho, Magada, and Nambale, respectively. Multiplex PCR on the pure cultures strongly supported that these isolates corresponded to X. oryzae pv. oryzicola. Two isolates, Ug_1 and Ug_14, were further subjected to partial DNA sequence analysis of the gyrB gene upon PCR amplification using the primers XgyrB1F and XgyrB1R (5). The 467-bp DNA sequence was identical to the gyrB sequences from the X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), and MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of strain Ug_1 was submitted to GenBank (KJ921786). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cultivars Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. For this purpose, bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water at a concentration of 1 × 108 CFU/ml. Bacterial suspensions were sprayed on leaves of rice seedlings. Four seedlings per accession and isolate were inoculated. Fifteen days after incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), inoculated leaves exhibited typical water-soaked lesions with yellow exudates that were similar to the symptoms seen in the fields. Re-isolation of the bacteria from the diseased leaves yielded colonies with the typical morphology of Xanthomonas. Multiplex PCR and sequence analysis of portions of the gyrB gene confirmed that these isolates are X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. One of the three isolates, Ug_1, has been deposited in the Collection Française de Bactéries Phytopathogènes (CFBP) as strain CFBP 8171 ( http://www.angers-nantes.inra.fr/cfbp/ ). Further surveys and strain collections in East and Central Africa will help assess the geographic distribution and importance of BLS. References: (1) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi.

On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Confirmation of Bacterial Leaf Streak Caused by Xanthomonas oryzae pv. oryzicola on Rice in Madagascar.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola is an important disease of rice. BLS is prevalent in Asia and West Africa, where it was first reported in Nigeria and Senegal in the early 1980s (4). Recently, molecular analysis of strains from Mali (2) and Burkina Faso (5) further confirmed the presence of BLS in West Africa. In Madagascar, BLS symptoms were first reported in the 1980s by Buddenhagen but the causal agent was not unequivocally determined (1). To confirm Buddenhagen's observations using modern molecular typing tools, we surveyed several rice fields in the Antananarivo and Antsirabe districts in March 2013. BLS symptoms were observed on cultivated Oryza sativa grown under both upland and lowland conditions, with a proportion of diseased individuals varying from 30% up to 80%. Symptomatic leaves presenting water-soaked lesions that developed into translucent, yellow streaks with visible exudates at the surface were sampled. One to four centimeter long pieces of diseased leaves were ground using the Qiagen TissueLyser system at 30 rps for 30 s (Qiagen, Courtaboeuf, France). The ground tissue was then macerated in 1 ml of sterile water for 1 h at 4°C. Non-diluted and 10-fold diluted tissue macerates were plated on semi-selective PSA medium (peptone 10 g/liter, sucrose 10 g/liter, glutamic acid 1 g/liter, bacto agar 16 g/liter, actidione 50 mg/liter, cephalexin 40 mg/liter, and kasugamycin 20 mg/liter) and incubated for 3 to 7 days at 28°C. Single, yellow, Xanthomonas-like colonies were isolated on non-selective PSA medium. Diagnostic multiplex PCR was performed on single colonies for pathovar identification (3). Five strains that produced three diagnostic bands corresponding to the X. oryzae pv. oryzicola pattern were further analyzed for pathogenicity on 3-week-old O. sativa cv. Nipponbare plants. Bacteria grown on PSA plates and adjusted to 1 × 108 CFU/ml were infiltrated into rice leaves with a needleless 1-ml syringe (2 × 3 infiltrations per plant and strain). Seven days after incubation in the greenhouse (27 ± 1°C with a 12-h photoperiod), inoculated leaves showed water-soaked lesions that produced yellow exudates corresponding to those initially observed in rice fields and observed for leaves challenged with the X. oryzae pv. oryzicola reference strain BLS256. Symptomatic leaf tissues were ground and plated on non-selective PSA medium, resulting in colonies with typical Xanthomonas morphology that were confirmed as X. oryzae pv. oryzicola by multiplex PCR typing (3), thus fulfilling Koch's postulates. Finally, the five strains were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F (5'-ACGAGTACAACCCGGACAA-3') and XgyrB1R (5'-CCCATCARGGTGCTGAAGAT-3'). The 743-bp partial gyrB sequences were 100% identical to the gyrB sequence of strain BLS256. As expected, the gyrB sequence of strains KACC10331, MAFF311018, and PXO99A of the X. oryzae pv. oryzae pathovar respectively showed nine, 16, and 10 mismatches in comparison to the Malagasy strains, thus further supporting that they belong to the pathovar oryzicola. References: (1) I. W. Buddenhagen. Int. Rice Comm. Newsl. 34:74, 1985. (2) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) D. O. Niño-Liu et al. Mol. Plant Pathol. 7:303, 2006. (5) I. Wonni et al. Plant Dis. 95:72, 2011.

High-Quality Draft Genome Sequence of Xanthomonas alfalfae subsp. alfalfae Strain CFBP 3836.

We report the high-quality draft genome sequence of Xanthomonas alfalfae subsp. alfalfae strain CFBP 3836, the causal agent of bacterial leaf and stem spot in lucerne (Medicago sativa). Comparative genomics will help to decipher the mechanisms provoking disease and triggering the defense responses of this pathogen of the model legume Medicago truncatula.

High-Quality Draft Genome Sequences of Xanthomonas axonopodis pv. glycines Strains CFBP 2526 and CFBP 7119.

We report here the high-quality draft genome sequences of two strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule on soybeans. Comparison of these genomes with those of phylogenetically closely related pathovars of Xanthomonas spp. will help to understand the mechanisms involved in host specificity and adaptation to host plants.

The role of bacterial pili in protein and DNA translocation.

Gram-negative bacteria have surface appendages that assemble via different secretion machineries. Recently, new experimental approaches have contributed to a better understanding of the molecular mechanisms of flagellar and pilus assembly, and protein secretion. These findings can be applied to plant pathogenic bacteria, which probably transfer effector proteins directly into their eukaryotic host cells. Here, it is suggested that assembly of Hrp pili occurs in the periplasm and that unfolded effector proteins attach to pilins within the pili, thus effecting protein translocation. A two-domain structure for the HrpA pilin from Pseudomonas syringae is also predicted.

Sucrose transport through maltoporin mutants of Escherichia coli.

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.

Structure and function of bacterial outer membrane proteins: barrels in a nutshell.

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

Extended sugar slide function for the periplasmic coiled coil domain of ScrY.

Several bacterial outer membrane proteins have a periplasmic extension whose structure and function remain elusive. Here, the structure/function relationship of the N-terminal periplasmic domain of the sucrose-specific outer membrane channel ScrY was investigated. Circular dichroism and analytical centrifugation demonstrated that the N-terminal domain formed a parallel, three-stranded coiled coil. When this domain was fused to the maltose-specific channel LamB, permeation of maltooligosaccharides in liposomes increased with increasing sugar chain length whereas wild-type LamB showed the opposite effect. Current fluctuation analysis demonstrated increased off-rates for sugar transport through the fusion protein. Moreover, equilibrium dialysis showed an affinity of sucrose for the isolated N-terminal peptide. Together these results demonstrate a novel function for coiled coil domains, operating as an extended sugar slide.

Sugar transport through maltoporin of Escherichia coli. Role of polar tracks.

The three-dimensional structure of the maltooligosaccharide specific outer membrane channel LamB of Escherichia coli complexed with sugar molecules revealed a hypothetical transport pathway. Sugars are supposed to slide over a stretch of aromatic residues facilitated by continuous making/breaking of hydrogen bonds between the hydroxyl groups of the sugars and charged amino acids, the "polar tracks." The effect of nine single and three multiple mutations in the polar track residues was investigated by current fluctuations, liposome swelling assays, and in vivo uptake of radiolabeled substrates. Additionally, sugar transport through wild-type LamB was investigated by current fluctuation analysis in water and deuterium. This way the effects on k(on) and k(off) could be investigated separately. Analyses of the various mutants revealed a strong effect on the k(on) values. Because steering to the binding site requires only a few interactions, consequently the loss of even one bond will have a strong effect. Deuterium experiments, which changed the characteristic of all hydrogen bonds, showed a strong effect on k(off) rates, because at this stage the sugar has numerous interactions with the channel. Furthermore, all the mutations induces a strong decrease of in vivo uptake of sugars. These results clearly demonstrate the importance of the polar track residues on both on and off rates in sugar transport and reveal a strong cooperative effect of hydrogen bond formation.

Structural and functional roles of the surface-exposed loops of the beta-barrel membrane protein OmpA from Escherichia coli.

The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel beta-barrel whose eight transmembrane beta-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest beta-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.

Membrane assembly of the Escherichia coli outer membrane protein OmpA: exploring sequence constraints on transmembrane beta-strands.

The eight-stranded antiparallel beta-barrel domain of the OmpA protein from Escherichia coli serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. Previous studies have shown that neither the periplasmic turns nor the surface-exposed loops contain topogenic information. Consequently, the question of whether any structural constraint is imposed onto individual transmembrane beta-strands is now addressed. To this end, amino acid sequences of beta-strands 4, 6 and 8 were randomized. In vivo membrane assembly of mutant proteins was assayed and 288 variants were sequenced. Three parameters were found to be important for efficient membrane assembly. (i) At least four of five randomized residues with side-chains pointing towards the lipid bilayer must be hydrophobic and none of the three central residues must be charged. (ii) Side-chains pointing into the beta-barrel interior must not be enlarged too much, possibly because of packing constraints. (iii) Proline residues are, in general, hardly tolerated in the transmembrane beta-strands.