Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter.

Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed "effectors," into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action.

Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Electroporation of functional bacterial effectors into mammalian cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Advancing the high throughput identification of liver fibrosis protein signatures using multiplexed ion mobility spectrometry.

Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While MS-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry separations with liquid chromatography and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the liquid chromatography--ion mobility spectrometry-MS platform analyzing blood serum samples from 60 postliver transplant patients with recurrent fibrosis progression and 60 nontransplant patients illustrates its potential utility for disease characterization.

Diverse secreted effectors are required for Salmonella persistence in a mouse infection model.

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

Multi-omic data integration links deleted in breast cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response.

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans.

The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.

New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium.

Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

RNA type III secretion signals that require Hfq.

Salmonella virulence is largely mediated by two type III secretion systems (T3SS) that deliver effector proteins from the bacterium to a host cell; however, the secretion signal is poorly defined. Effector N termini are thought to contain the signal, but they lack homology, possess no identifiable motif, and adopt intrinsically disordered structures. Alternative studies suggest that RNA-encoded signals may also be recognized and that they can be located in the 5' untranslated leader sequence. We began our study by establishing the minimum sequence required for reporter translocation. Untranslated leader sequences predicted from 42 different Salmonella effector proteins were fused to the adenylate cyclase reporter (CyaA'), and each of them was tested for protein injection into J774 macrophages. RNA sequences derived from five effectors, gtgA, cigR, gogB, sseL, and steD, were sufficient for CyaA' translocation into host cells. To determine the mechanism of signal recognition, we identified proteins that bound specifically to the gtgA RNA. One of the unique proteins identified was Hfq. Hfq had no effect upon the translocation of full-length CigR and SteD, but injection of intact GtgA, GogB, and SseL was abolished in an hfq mutant, confirming the importance of Hfq. Our results demonstrated that the Salmonella pathogenicity island 2 (SPI-2) T3SS assembled into a functional apparatus independently of Hfq. Since particular effectors required Hfq for translocation, Hfq-RNA complexes may participate in signal recognition.

Studying Salmonellae and Yersiniae host-pathogen interactions using integrated 'omics and modeling.

Salmonella and Yersinia are two distantly related genera containing species with wide host-range specificity and pathogenic capacity. The metabolic complexity of these organisms facilitates robust lifestyles both outside of and within animal hosts. Using a pathogen-centric systems biology approach, we are combining a multi-omics (transcriptomics, proteomics, metabolomics) strategy to define properties of these pathogens under a variety of conditions including those that mimic the environments encountered during pathogenesis. These high-dimensional omics datasets are being integrated in selected ways to improve genome annotations, discover novel virulence-related factors, and model growth under infectious states. We will review the evolving technological approaches toward understanding complex microbial life through multi-omic measurements and integration, while highlighting some of our most recent successes in this area.

Unexpected diversity of signal peptides in prokaryotes.

UNLABELLED: Signal peptides are a cornerstone mechanism for cellular protein localization, yet until now experimental determination of signal peptides has come from only a narrow taxonomic sampling. As a result, the dominant view is that Sec-cleaved signal peptides in prokaryotes are defined by a canonical AxA motif. Although other residues are permitted in the motif, alanine is by far the most common. Here we broadly examine proteomics data to reveal the signal peptide sequences for 32 bacterial and archaeal organisms from nine phyla and demonstrate that this alanine preference is not universal. Discoveries include fundamentally distinct signal peptide motifs from Alphaproteobacteria, Spirochaetes, Thermotogae and Euryarchaeota. In these novel motifs, alanine is no longer the dominant residue but has been replaced in a different way for each taxon. Surprisingly, divergent motifs correlate with a proteome-wide reduction in alanine. Computational analyses of ~1,500 genomes reveal numerous major evolutionary clades which have replaced the canonical signal peptide sequence with novel motifs. IMPORTANCE: This article replaces a widely held general model with a more detailed model describing phylogenetically correlated variation in motifs for Sec secretion.

A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions.

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling.

Genome-scale modeling of light-driven reductant partitioning and carbon fluxes in diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142.

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

Identification of c-type heme-containing peptides using nonactivated immobilized metal affinity chromatography resin enrichment and higher-energy collisional dissociation.

The c-type cytochromes play essential roles in many biological activities of both prokaryotic and eukaryotic cells, including electron transfer, enzyme catalysis, and induction of apoptosis. We report a novel enrichment strategy for identifying c-type heme-containing peptides that uses nonactivated IMAC resin. The strategy demonstrated at least 7-fold enrichment for heme-containing peptides digested from a cytochrome c protein standard, and quantitative linear performance was also assessed for heme-containing peptide enrichment. Heme-containing peptides extracted from the periplasmic fraction of Shewanella oneidensis MR-1 were further identified using higher-energy collisional dissociation tandem mass spectrometry. The results demonstrated the applicability of this enrichment strategy to identify c-type heme-containing peptides from a highly complex biological sample and, at the same time, confirmed the periplasmic localization of heme-containing proteins during suboxic respiration activities of S. oneidensis MR-1.

Extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics.

The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR-1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.

Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.

Mapping the subcellular proteome of Shewanella oneidensis MR-1 using sarkosyl-based fractionation and LC-MS/MS protein identification.

A simple and effective subcellular proteomic method for fractionation based on osmotic lysis, differential centrifugation, and Sarkosyl solubilization was applied to the Gram-negative bacterium Shewanella oneidensis to gain insight into its subcellular architecture. Global differences in bacterial cytoplasm, inner membrane, periplasm, and outer membrane protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution liquid chromatography-tandem mass spectrometry. Proteins predicted to be localized to each subcellular fraction were enriched approximately 2-fold (on average) in each fraction compared to crude cell lysates. In addition, the Sarkosyl solubilization method facilitated separation of the inner and outer membranes, making the procedure amenable for effective probing of the subcellular proteome of Gram-negative bacteria via liquid chromatography-tandem mass spectrometry. With 40% of the observable proteome represented, this study provides extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other Gram-negative bacteria.

Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling.

We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope (18)O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a Gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level (16)O and (18)O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in Delta gspD mutant cells of many outer membrane proteins including the outer membrane c-type cytochromes OmcA and MtrC, in agreement with a previous report that these proteins are substrates of the type II secretion system.

Roles of RseB, sigmaE, and DegP in virulence and phase variation of colony morphotype of Vibrio vulnificus.

Vibrio vulnificus is an estuarine bacterium capable of causing serious and often fatal wound infections and primary septicemia. We used alkaline phosphatase insertion mutagenesis to identify genes necessary for the virulence of this pathogen. One mutant had an in-frame fusion of 'phoA to the gene encoding RseB, a periplasmic negative regulator of the alternative sigma factor sigma(E). sigma(E) controls an extensive regulon involved in responding to cell envelope stresses. Colonies of the rseB mutant were less opaque than wild-type colonies and underwent phase variation between translucent and opaque morphologies. rseB mutants were attenuated for virulence in subcutaneously inoculated iron-dextran-treated mice. To obtain insight into the role of rseB and the extracytoplasmic stress response in V. vulnificus, mutants with defined mutations in rseB and two important members of the extracytoplasmic stress regulon, rpoE and degP, were constructed for analysis of virulence, colony morphology, and stress-associated phenotypes. Deletion of rseB caused reversible phase variation in the colony morphotype that was associated with extracellular polysaccharides. Translucent and transparent morphotype strains were attenuated for virulence. rpoE and degP deletion mutants were sensitive to membrane-perturbing agents and heat but were not significantly attenuated for V. vulnificus virulence in mice. These results reveal complex relationships between regulation of the extracytoplasmic stress response, exopolysaccharides, and the virulence of V. vulnificus.

USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus.

Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.