Outer membrane protein genes and their small non-coding RNA regulator genes in Photorhabdus luminescens

INTRODUCTION: Three major outer membrane protein genes of Escherichia coli, ompF, ompC, and ompA respond to stress factors. Transcripts from these genes are regulated by the small non-coding RNAs micF, micC, and micA, respectively. Here we examine Photorhabdus luminescens, an organism that has a different habitat from E. coli for outer membrane protein genes and their regulatory RNA genes. RESULTS: By bioinformatics analysis of conserved genetic loci, mRNA 5'UTR sequences, RNA secondary structure motifs, upstream promoter regions and protein sequence homologies, an ompF -like porin gene in P. luminescens as well as a duplication of this gene have been predicted. Gene loci for micF RNA, as well as OmpC protein and its associated regulatory micC RNA, were not found. Significantly, a sequence bearing the appropriate signatures of the E. coli micA RNA was located. The ompA homolog was previously annotated in P. luminescens. CONCLUSION: Presence of an ompF-like porin in P. luminescens is in keeping with the necessity to allow for passage of small molecules into the cell. The apparent lack of ompC, micC and micF suggests that these genes are not essential to P. luminescens and ompC and micF in particular may have been lost when the organism entered its defined life cycle and partially protected habitat. Control of porin gene expression by RNA may be more prevalent in free- living cells where survival is dependent on the ability to make rapid adjustments in response to environmental stress. Regulation of ompA by micA may have been retained due to a necessity for ompA control during one or both stages of the P. luminescens life cycle. REVIEWERS: This article was reviewed by Tal Dagan (nominated by Dan Graur), Mikhail Gelfand and Anna Gerasimova (nominated by Mikhail Gelfand) and J Peter Gogarten. OPEN PEER REVIEW: Reviewed by Tal Dagan (nominated by Dan Graur), Mikhail Gelfand and Anna Gerasimova (nominated by Mikhail Gelfand) and J Peter Gogarten. For the full reviews, please go to the Reviewers' comments section

Trends of the major porin gene (ompF) evolution

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species

An improved method for surface immobilisation of RNA: application to small Non-Coding RNA - mRNA pairing

Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3' end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3' end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA)-mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms

Contribution to unveiling the roles played by small non-coding RNAs in the biology and pathogenesis of Burkholderia cepacia complex bacteria

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2017The development of high-throughput sequencing techniques and continued decrease of associated costs, together with advances in bioinformatics and increasing availability of powerful software for nucleotide and amino acid sequence analyses contributed to the exponential increase of available microbial genomes. These developments have unveiled several novel sequences presenting a size within the range of 50-500 nucleotides (nt) and encoding the now called small non-coding regulatory RNAs (sRNAs). These sRNAs are frequently encoded in intergenic regions, often partially overlapping with the 5’ or 3’ untranslated regions of the vicinal genes or annotated-opening reading frames (ORFs). The current known function for some sRNAs comprises the gene expression regulation through interference RNA mechanisms, mainly at the posttranscriptional level. Bacterial sRNAs may regulate their targets (messenger RNAs) through positive or negative regulation mechanisms. To carry out a negative regulation, the sRNA base pairs with its target in the region containing the initiation codon and/or the ribosome-binding site (RBS), precluding the ribosome binding and consequently preventing the mRNA translation. The Burkholderia cepacia (Bcc) complex comprises nowadays 20 validated and phylogenetically-related species of Gram-negative bacteria. These bacteria are phenotypically similar and genotypically distinct, being widely distributed in different ecological niches such as soil, water, plants, animals and in humans. Some Bcc bacteria have potential application as bioremediation and biocontrol/biopesticides agents due to their unusual metabolic abilities, which include xenobiotics metabolism, production of antifungal compounds and promotion of plant growth. However, Burkholderia species have emerged in 1980s as important opportunistic pathogens, especially to cystic fibrosis (CF) patients. Bcc bacteria have gained the attention of medical and scientific community because they can cause in CF patients a rapid evolving clinical state known as the cepacia syndrome, which can cause a fast and necrotizing pneumonia, septicemia and ultimately results in the patient early death. Bcc bacteria produce a wide variety of virulence factors, possessing intrinsic resistance mechanisms to different antibiotics which lead to a difficult eradication. Over the last years, some Bcc species have also been recognized as emerging nosocomial pathogenic agents in hospitalized non-CF patients, particularly in cancer patients. To identify potential genes involved in the Bcc bacteria virulence, researchers from iBB (Instituto de Bioengenharia e Biociências) prepared mutant libraries derived from B. contaminans IST408 and B. cenocepacia J2315 by random plasposon mutagenesis. This work aims to characterize a mutant derived from B. cenocepacia J2315, identified in an attenuated virulence screening using as model of infection the nematode Caenorhabditis elegans. This mutant, B. cenocepacia SJ2, contains the plasposon inserted in the intergenic region of B. cenocepacia J2315 chromosome 1, located between the coding gene of DNA gyrase subunit A (GyrA) enzyme and the coding gene of an outer membrane protein of the OmpA family. Bioinformatic and Southern blot analyses allowed the identification of the plasposon insertion in the mutant B. cenocepacia SJ2. The plasposon is located 8 nucleotides (nt) after the predicted 5’ UTR beginning of the B. cenocepacia J2315 OmpA-like protein. Therefore, the presence and functionality of the transcript ompA were evaluated by RT-PCR and Western blot, respectively. The results obtained indicated that the plasposon insertion did not affect ompA mRNA transcription or functionality. Bioinformatic analyses also led to the identification of a possible opening reading frame (ORF) in the complementary strand (named ORF3) and encoding a 93 amino acids protein. The presence of this ORF was supported by Northern blot assays previously performed. These assays allowed the identification of a transcript from the complementary strand. However, the amplification of complementary DNA ends (5’ and 3’ RACE) using specific primers for ORF3 region demonstrated that the transcript in study has a 5’ end with a lower size than the expected transcript of the potential protein (ORF3). A transcript containing approximately 179 base pairs and non-interrupted by the plasposon was identified. In the presence of these results, primers for the potential DNA sequence of ORF3 were designed and the results showed that this is the region interrupted by the plasposon. Due to the absence of initiation and termination codons in the region closer to the 179 base pairs transcript, it was hypothesized that the transcript corresponds to a sRNA (MavA). In this work, the presence of two genetic elements, a sRNA and a protein, in the intergenic region of B. cenocepacia J2315 chromosome 1 is described. The coding sequences of both the sRNA and protein are overlapped in, at least, 100 nucleotides. Bioinformatic analysis of the genetic elements showed that they are conserved among the Burkholderia genus. Moreover, the sequence of ORF3 protein also showed to be identical to the sequence of one protein encoded by an annotated ORF in the B. multivorans genome and to five proteins of B. pseudomallei. The assays performed with the strain containing the ORF3 interrupted by the plasposon revealed the involvement of this protein in the resistance of B. cenocepacia J2315 strain to heat-shock stress (50 ºC), susceptibility to the detergent sodium dodecyl sulphate (SDS), biofilms formation, cellular hydrophobicity and antibiotics resistance (imipenem, ceftazidime and tetracycline). In general, these results suggest that this protein is likely involved in the maintenance of the outer membrane integrity and virulence of B. cenocepacia J2315 towards the nematode C. elegans. MavA sRNA identified in the work herein presented was predicted to be a functional homologue of IstR-2 sRNA of Escherichia coli K-12 MG1655. Preliminary results of the MavA sRNA overexpression characterization indicate a possible direct or indirect role in the overexpression of ribosomal protein S12. Phenotypic analysis also showed the involvement in the swimming motility of B. cenocepacia J2315 strain and in resistance to thermal (50 ºC) stress. In addition, this sRNA is not involved in the virulence of B. cenocepacia J2315 towards the C. elegans nematode. Overall, the results presented in this study contribute to a better knowledge of the intergenic region under study and to the identification of a putative ORF and a sRNA, contributing to a better understanding of the biology of Bcc bacteria and the role of sRNAs in the regulation of the expression of putative virulence factors. The identification of MavA sRNA and ORF3 protein highlight the importance of these studies in identifying genetic elements that might be exploited as targets for the development of effective treatments to the B. cenocepacia J2315 strain infections.O recente desenvolvimento de técnicas de elevado rendimento para sequenciar genomas e a diminuição contínua do custo associado, conjuntamente com os avanços na bioinformática e a disponibilidade de número crescente de ferramentas bioinformáticas para análise de sequências nucleotídicas e aminoacídicas, tornou possível o aumento exponencial dos genomas microbianos disponíveis. Consequentemente, têm vindo a ser descobertos diversos pequenos transcritos com ação regulatória denominados de pequenos RNAs reguladores não codificantes (sRNAs), com tamanhos compreendidos entre 50-500 nucleótidos. Estes sRNAs têm sido identificados essencialmente em regiões intergénicas, junto a genes ou grelhas de leitura (ORFs) anotadas. A função conhecida de um grupo importante desses sRNAs consiste na regulação da expressão génica, principalmente ao nível pós-transcricional, por mecanismos de interferência de RNA. Os sRNAs bacterianos podem exercer nos seus alvos (RNAs mensageiros) uma regulação positiva ou negativa. No caso de ser exercida uma regulação negativa o sRNA estabelece com o seu alvo um emparelhamento na região que contém o codão de iniciação e/ou a região de ligação do ribossoma (RBS), tornando este local inacessível para a ligação do ribossoma e impedindo, assim, a tradução do mRNA. O complexo Burkholderia cepacia (Bcc) é atualmente constituído por 20 espécies de bactérias Gram-negativas validadas e filogeneticamente próximas. As bactérias que compõem este grupo são fenotipicamente semelhantes e genotipicamente distintas, podendo ser isoladas de várias fontes. São exemplo de fontes o solo, a água, a rizosfera de plantas, animais e Humanos. Devido às suas capacidades metabólicas invulgares, algumas estirpes do Bcc apresentam um elevado potencial de aplicação ao nível do biocontrolo, biorremediação e agricultura, pois são capazes de metabolizar xenobióticos, produzir compostos com atividade antifúngica e promover o crescimento de plantas. Contudo, na década de 80 as bactérias incluídas no género Burkholderia, incluindo as englobadas no complexo Bcc, emergiram como agentes patogénicos oportunistas em indivíduos com fibrose quística (FQ). O facto de as bactérias deste complexo produzirem vários fatores de virulência, apresentarem mecanismos de resistência a um largo espetro de antibióticos sendo difíceis de erradicar e de causarem em doentes com FQ infeções por vezes acompanhadas por um estado clínico de evolução rápida que inclui pneumonia necrotizante e septicémia, levando à morte do doente – síndrome cepacia -, faz com que tenham merecido uma elevada atenção da comunidade médica e científica. Acresce que algumas espécies do Bcc têm vindo a ser reconhecidas nos últimos anos como sendo agentes patogénicos nosocomiais emergentes em doentes hospitalizados, principalmente em doentes oncológicos. Com o objetivo de identificar possíveis genes envolvidos na virulência de bactérias do Bcc, investigadores do iBB (Instituto de Bioengenharia e Biociências) construíram bibliotecas de mutantes de B. contaminans IST408 e B. cenocepacia J2315 utilizando plasposões. As bibliotecas de mutantes foram rastreadas tendo como objetivo a identificação de mutantes que exibiam virulência atenuada, utilizando como modelo de infeção o nemátodo Caenorhabditis elegans. O trabalho aqui apresentado teve como objetivo a caracterização de um mutante identificado no rastreio acima mencionado, contendo um plasposão inserido na região intergénica do cromossoma 1 de B. cenocepacia J2315, localizada entre o gene codificante da subunidade A da enzima ADN girase (GyrA) e o gene codificante de uma proteína da membrana externa do tipo A (OmpA). Através de análises bioinformáticas e experimentais, foi possível localizar no mutante B. cenocepacia SJ2 a inserção do plasposão 8 nucleótidos (nt) após o início da região 5’ não traduzida (5’ UTR) do gene que codifica a proteína do tipo OmpA. Neste sentido, foram avaliados a presença e a funcionalidade do transcrito ompA por ensaios de RT-PCR e Western blot, respetivamente. Os resultados obtidos confirmaram que a inserção do plasposão não afetou a transcrição nem a funcionalidade do transcrito correspondente ao gene ompA. As análises bioinformáticas realizadas permitiram ainda a identificação de uma possível grelha de leitura aberta (ORF) na cadeia complementar da região intergénica (designada de ORF3), codificando para uma proteína de aproximadamente 93 aminoácidos. A confirmação da presença desta ORF foi suportada por ensaios de Northern blot realizados anteriormente e que permitiram a identificação de um transcrito a partir da cadeia complementar. No entanto, análises de amplificação em cadeia pela polimerase (PCR) das extremidades do ADN complementar (5’ e 3’ RACE) com sequências oligonucleotídicas iniciadoras específicas para a região da ORF3 demonstraram que o transcrito em causa possui uma extremidade 5’ menor que o esperado para o transcrito da possível proteína (ORF3), permitindo assim a identificação de um transcrito com cerca de 179 pares de bases que se verificou não ser interrompido pelo plasposão. Deste modo, tendo em conta os resultados aqui apresentados foram desenhadas sequências oligonucleotídicas iniciadoras específicas para a sequência de ADN da possível ORF3, tendo-se verificado que esta era a região interrompida pelo plasposão. Devido à ausência de codões de iniciação e de terminação na região próxima do transcrito de 179 pares de base, colocou-se a hipótese deste transcrito corresponder a outro elemento genético, nomeadamente um sRNA, que se denominou de MavA. Neste trabalho é descrita a existência de dois elementos genéticos na região intergénica do cromossoma 1 de B. cenocepacia J2315, um sRNA e um gene que codifica uma proteína, sendo que as sequências codificantes de ambos encontram-se parcialmente sobrepostas em, pelo menos, 100 nucleótidos. As análises bioinformáticas realizadas dos elementos genéticos permitiram a sua identificação como sendo ambos conservados no género Burkholderia. Além disso, demonstraram também que a proteína ORF3 é idêntica à sequência de uma proteína codificada por uma ORF anotada no genoma de B. multivorans e à de cinco proteínas de B. pseudomallei. Ensaios realizados com a estirpe contendo o plasposão a interromper a ORF3 demonstraram que esta proteína está envolvida na resistência da estirpe B. cenocepacia J2315 a choque térmico (50 ºC), suscetibilidade à presença do detergente dodecil sulfato de sódio (SDS), capacidade para formação de biofilmes e hidrofobicidade das células, bem como na resistência a antibióticos (imipeneme, ceftazidima e tetraciclina). De um modo geral, estes resultados apontam para que esta proteína esteja envolvida na manutenção da integridade da membrana externa e virulência da estirpe B. cenocepacia J2315 para o nemátodo C. elegans. O sRNA MavA identificado neste trabalho foi bioinformaticamente previsto como tendo como seu homólogo funcional o sRNA IstR-2 de Escherichia coli K-12 MG1655. Os resultados preliminares da caracterização do sRNA MavA através da sua sobrexpressão demonstram um possível envolvimento direto ou indireto na sobrexpressão da proteína ribossomal S12. As análises fenotípicas mostraram ainda o seu envolvimento na mobilidade da estirpe B. cenocepacia J2315 por swimming e na resistência ao choque térmico (50 ºC). Os resultados obtidos, baseados em ensaios de morte lenta do nemátodo C. elegans sugerem que este sRNA não está envolvido na virulência de B. cenocepacia J2315 neste modelo animal de infeção. Os resultados obtidos no presente trabalho contribuem para um conhecimento mais aprofundado da região intergénica estudada, tendo permitido identificar 2 elementos genéticos parcialmente sobrepostos que codificam uma proteína e um sRNA. Este trabalho constitui assim um contributo para o melhor conhecimento da biologia das bactérias do complexo Bcc e do papel dos sRNAs na regulação da expressão de fatores de virulência. A identificação do sRNA MavA e da proteína ORF3 realça a importância deste tipo de estudos na identificação de elementos genéticos que possam ser explorados como alvos para o desenvolvimento de estratégias terapêuticas mais eficazes com vista ao tratamento das infeções causadas pela estirpe B. cenocepacia J2315

The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium

Dissertation presented to obtain the Ph.D degree in BiologyRNAs are important effectors in the process of gene expression. In bacteria, the levels of the transcripts have to be rapidly adjusted in response to constantly changing environmental demands. The cellular concentration of a given RNA is the result of the balance between its synthesis and degradation. RNA degradation is a complex process encompassing multiple pathways. Ribonucleases are the enzymes that directly process and degrade RNA transcripts, regulating their cellular amounts. The rate at which RNA decay occurs depends on the availability of ribonucleases and their specificities according to the sequence and/or the structural elements of the RNA molecule. Several other factors modulate RNA degradation, namely polyadenylation, which plays a multifunctional role in RNA metabolism. Additionally, small non-coding RNAs are crucial regulators of gene expression, and can directly modulate the stability of their mRNA targets. In many cases this regulation is dependent on Hfq, an RNA binding protein which can act in concert with polyadenylation enzymes and is often necessary for the activity of the sRNAs.(...

Principles of RNA-based gene expression control in Vibrio cholerae

Post-transcriptional control of gene expression by small regulatory RNAs (sRNAs) is a widespread regulatory principle among bacteria. The sRNAs typically act in concert with RNA binding proteins such as the RNA chaperone Hfq to bind mRNA targets via imperfect base pairing. They affect translation initiation and/or transcript stability. Additionally, sRNAs can influence transcription termination of their targets or function indirectly as so-called sponges for other sRNAs. Regulation often involves the major endoribonuclease RNase E, which contributes to both sRNA biosynthesis and function. In the first part of this thesis, we globally identified RNase E cleavage sites in the major human pathogen Vibrio cholerae by employing TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq). We validated the involvement of RNase E in the synthesis and maturation of several previously uncharacterized sRNAs. Two examples, OppZ and CarZ, were chosen for further study due to their unique regulatory mechanism. They are processed from the 3’ untranslated regions (3’ UTR) of the oppABCDF and carAB operons, respectively, and subsequently target mRNAs transcribed from the very same operons by binding to base pairing sites upstream of the second (oppB) or first (carA) cistrons. This leads to translational inhibition and triggers premature transcription termination by the termination factor Rho, thereby establishing an autoregulatory feedback loop involving both the protein-coding genes and the processed sRNAs. In the case of OppZ, the regulation is limited to the oppBCDF part of the operon in a discoordinate fashion due to the position of the OppZ base pairing site. This mechanism of target regulation by Opp and CarZ represents the first report of an RNA-based feedback regulation that does not rely on additional transcription factors. The second study included in the thesis characterizes two sRNAs involved in the envelope stress response (ESR) of V. cholerae. Misfolded outer membrane proteins (OMPs) induce the sigmaE-dependent transcriptional activation of the sRNAs MicV and VrrA, which reduce membrane stress by repressing the mRNAs of several OMPs and other abundant membrane protein. MicV and VrrA share a conserved seed region with their functionally analogous counterpart from Escherichia coli, RybB, indicating that this seed sequence might represent a universally functional RNA domain. To study the involvement of this seed domain in the ESR in an unbiased fashion, we constructed a complex library of artificial sRNAs and performed laboratory selection experiments under membrane-damaging conditions. We isolated the most highly enriched sRNA variants and indeed discovered a strong enrichment of the conserved seed-pairing domain. We were able to pinpoint the repression of ompA as the key factor responsible for the sRNA-mediated resistance to ethanol-induced membrane damage. Taken together, this thesis expanded the knowledge on the mechanisms of sRNA-dependent gene regulation by reporting a novel autoregulatory feedback loop. Additionally, it introduced a synthetic sRNA library as a tool to study complex microbial phenotypes and their underlying sRNA-target interactions

Antibiotic Stress, Genetic Response and Altered Permeability of E. coli

BACKGROUND: Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure

Export and Regulatory Properties of MalE Hybrid Proteins in <i>Escherichia coli</i>

The maltose binding protein (MalE) of Escherichia coli is a secreted 370 residue polypeptide which serves as the periplasmic receptor for high-affinity membrane transport of maltose and maltodextrins. Cells tolerate synthesis and translocation across the plasma membrane of large quantities of MalE. Similarly, the outer membrane protein Omp A is not toxic when expressed in physiological amounts. Expression of an ompA-malE hybrid gene consisting of the first 274 residues of OmpA and the last 251 residues of MalE was, however, toxic, although synthesis of the OmpA part alone was not. To examine the effect of synthesis of the MalE part of the hybrid, the malE fragment was cloned into a vector where it was preceded by the gene coding for the OmpA signal peptide and was inducible by isopropy Ithiogalactoside. Upon induction, a polypeptide of the expected size was made, but expression of the truncated MalE turned out to be even more toxic than that of the OmpA-MalE hybrid. The MalE fragment was exported to the periplasm and did not interfere with the export of other secreted polypeptides. A search for suppressors of this toxicity was performed. In a chromosomal gene bank of E. coli, such a suppressor was found which coded for a 102 A-terminal residue fragment of the 217 residue protein NlpE (new outer membrane lipoprotein). NlpE is known to combat the toxicity of the cytosolic ß-galactosidase (LacZ) when artificially exported to the periplasm. This suppression is achieved by activation of the Cpx two-component signal transduction pathway controlling expression of the periplasmic protease DegP. Increased synthesis of DegP caused degradation of LacZ. The same mechanism appears to operate for the degradation of the MalE fragment. In a degP background, the NlpE fragment could not suppress toxicity. Mutants (cpxA*) exist which, without any signal, produced increased levels of DegP. In a cpxA* strain, the MalE fragment was no longer toxic. The toxicity caused by the MalE fragment can still not entirely be explained. However, several lines of evidence, such as the expression of the periplasmic spheroplast protein Y in cells producing MalE251, suggest that the most likely explanation for the lethality is a defect in the cell wall of induced cells