Colony Spreading Research Articles

Modulation of Staphylococcus aureus spreading by water.

Staphylococcus aureus is known to spread rapidly and form giant colonies on the surface of soft agar and animal tissues by a process called colony spreading. So far, the mechanisms underlying spreading remain poorly understood. This study investigated the spreading phenomenon by culturing S. aureus and its mutant derivatives on Tryptic Soy Agarose (TSA) medium. We found that S. aureus extracts water from the medium and floats on water at 2.5 h after inoculation, which could be observed using phase contrast microscopy. The floating of the bacteria on water could be verified by confocal microscopy using an S. aureus strain that constitutively expresses green fluorescence protein. This study also found that as the density of bacterial colony increases, a quorum sensing response is triggered, resulting in the synthesis of the biosurfactants, phenolic-soluble modulins (PSMs), which weakens water surface tension, causing water to flood the medium surface to allow the bacteria to spread rapidly. This study reveals a mechanism that explains how an organism lacking a flagellar motor is capable of spreading rapidly on a medium surface, which is important to the understanding of how S. aureus spreads in human tissues to cause infections.

Biofilm formation and cell surface properties of Staphylococcus aureus isolates from various sources.

This study investigated biofilm formation, cell surface hydrophobicity, colony spreading, and slime production for 112 Staphylococcus aureus strains isolated from various sources (leaf vegetables, pea leaf, perilla leaf, Kim-bab, person, and animal). When biofilm formation was classified by origin, S. aureus isolated from animal origin showed a significantly higher level of biofilm formation than others (p≤0.05). When S. aureus groups with different levels of biofilm formation (very strong, strong, moderate, and weak) were evaluated for the correlation with cell surface properties, there was a positive correlation between biofilm formation and hydrophobicity (r=0.926). Biofilm formation and colony spreading on tryptic soy broth (without dextrose) also showed positive correlation (r=0.863). In contrast, biofilm formation and slime production were negatively correlated (r=-0.973). Based on these results, the biofilm forming ability of S. aureus differs depending on their origin and might be affected by cell surface properties such as cell surface hydrophobicity.

Impact of psm-mec in Methicillin-Resistant Staphylococcus aureus (ST764) Strains Isolated from Keratitis Patients.

Staphylococcus aureus is a predominant pathogen in keratitis, and the rate of methicillin-resistant S. aureus (MRSA) is increasing. In our previous study, genotypes of MRSA isolates from keratitis cases were classified into ST5 or ST764 lineage by multi-locus sequence typing. In this study, we examined the virulence properties of these MRSA keratitis isolates and its virulence determinants. There was no difference in the prevalence of virulence genes, such as adhesion and toxins, between ST5 and ST764 isolates. All ST5 isolates carried the intact psm-mec gene, which suppresses exotoxin production and colony spreading, but promotes biofilm formation. In contrast, all ST764 isolates had one point mutation in the psm-mec gene. Biofilm production in ST5 isolates was significantly higher than that in ST764 isolates, whereas colony spreading, hemolytic activity, and production of alpha-phenol-soluble modulins were higher in ST764 than in ST5 isolates. The toxicity of ST764 supernatants to corneal epithelial cells was higher than that of ST5 supernatants. These results suggest that the point mutation in the psm-mec gene contributes to the difference in virulence properties between ST5 and ST764 isolates in MRSA keratitis.

Phenotypic Characteristics of Vancomycin-Non-Susceptible Staphylococcus aureus.

Background:Staphylococcus aureus, with reduced vancomycin susceptibility, is probably under the regulation of several genes and various express phenotypes.Objectives:This study aimed to investigate the phenotypic differences between vancomycin-susceptible S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) isolates.Materials and Methods:A total of 130 methicillin-resistant S. aureus (MRSA) isolates were studied, including 49 VSSA, 28 hVISA, and 5 VISA isolates from blood cultures and 48 isolates (two VSSA, six hVISA, and 40 VISA) derived in vitro (laboratory-induced/sub-passaged). Their phenotypes were examined using a coagulase tube test, colony spreading on soft agar, and urease activity. The SCCmec and agr typing were performed using multiplex PCR.Results:Most of the MRSA isolates were SCCmec III-agr I (84.5%), followed by SCCmec II-agr II (11.8%). The average plasma coagulation time of vancomycin-non-susceptible isolates was longer than that of the susceptible isolates (12 vs. 2.6 hours). Four hVISA (P = 0.023) and nine VISA (P < 0.001) isolates yielded a negative coagulase test after 24-hour incubation. The percentage of VSSA isolates showing non-spreading colonies (accessory gene regulator (agr) dysfunction) was significantly lower than in the VISA group (P = 0.013), but no significant difference was found between VSSA and hVISA. The VISA group showed higher urease activity than that of the VSSA and hVISA groups (P = 0.002).Conclusions:There were diverse phenotypic changes among vancomycin-non-susceptible S. aureus isolates. This may be due to the variety of related regulatory systems. The diversity of phenotypic expression may result in its misidentification in routine laboratory checks.

The Fossil and the Photograph: Red Cloud, Prehistoric Media, and Dispossession in Perpetuity

Two late-nineteenth-century media technologies purportedly recording the evolutionary past—the fossil and the photograph—helped naturalize the spread of settler colonialism across the Northern Plains. Settler colonial biopower works in the spaces in between life and death to dispossess indigenous peoples of their land in perpetuity, seizing prehistory simultaneously with the present and the future. I uncover how Oglala leader Red Cloud drew on indigenous epistemologies of both fossils and photographs to negotiate the nuances of temporal and material dispossession, particularly through his work as a photographic subject. Red Cloud’s interventions with paleontologists and photographers illuminate how new approaches to media history that question the biopolitical divide between the organic and inorganic could be important components of denaturalizing settler colonialism.

From a thin film model for passive suspensions towards the description of osmotic biofilm spreading

Biofilms are ubiquitous macro-colonies of bacteria that develop at various interfaces (solid-liquid, solid-gas or liquid-gas). The formation of biofilms starts with the attachment of individual bacteria to an interface, where they proliferate and produce a slimy polymeric matrix - two processes that result in colony growth and spreading. Recent experiments on the growth of biofilms on agar substrates under air have shown that for certain bacterial strains, the production of the extracellular matrix and the resulting osmotic influx of nutrient-rich water from the agar into the biofilm are more crucial for the spreading behaviour of a biofilm than the motility of individual bacteria. We present a model which describes the biofilm evolution and the advancing biofilm edge for this spreading mechanism. The model is based on a gradient dynamics formulation for thin films of biologically passive liquid mixtures and suspensions, supplemented by bioactive processes which play a decisive role in the osmotic spreading of biofilms. It explicitly includes the wetting properties of the biofilm on the agar substrate via a disjoining pressure and can therefore give insight into the interplay between passive surface forces and bioactive growth processes.

Fibrin gel as a scaffold for skin substitute – production and clinical experience.

The purpose of this study was to create a fibrin-based human skin substitute in vitro with epidermal and dermal component and to assess its healing potential in deep partial and full thickness burns. Fibrin scaffolds were prepared from commercial fibrin glue kits. Human fibroblasts were cultured in fibrin gel. Human keratinocytes were seeded on the top of the gel. Viability of cells was determined fluorimetrically. Scanning electron microscope and immunocytochemistry analysis of cultured cells were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for skin substitutes. Progress of healing was documented using visual estimation and photos. Scanning electron microscope images showed good cell attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaff old. Immunofluorescent staining of cell cultures on fibrin scaffold showed expression of vimentin, a marker of fibroblast cells, cytokeratin 19, a marker of epithelial stem cells, as well as involucrin, a marker of differentiated keratinocytes. Clinical results clearly showed that appearance of the skin did not differ significantly from the areas of transplanted skin using split-thickness skin graft techniques. In conclusion, using these fibrin-cultured autografts on massive full-thickness burn resulted in good healing.

A small periplasmic protein essential for Cytophaga hutchinsonii cellulose digestion.

Cytophaga hutchinsonii is a gliding cellulolytic bacterium that is ubiquitously distributed in soil. The mechanism by which C. hutchinsonii achieves cellulose digestion, however, is still largely unknown. In this study, we obtained a C. hutchinsonii mutant that was defective in utilizing filter paper or Avicel as the sole carbon source by transposon mutagenesis. The interrupted gene locus, CHU_2981, encodes a hypothetical protein with only 130 amino acids. Cell fractionation and western blot detection of CHU_2981 fused with a C-terminal green fluorescence protein (GFP) indicated that CHU_2981 is located in the periplasm. The CHU_2981-disrupted mutant cells exhibited a significant growth defect on Avicel but not on glucose and cellobiose. The absence of CHU_2981 also resulted in a significant defect in colony spreading and individual cell motility compared to wild-type cells. Further analysis demonstrated that the CHU_2981-disrupted mutant cells exhibited a different profile of cellulose-absorbed outer membrane proteins from that of wild-type cells, in which protein varieties and amounts were markedly decreased. Our results showed that CHU_2981, the periplasmic non-cellulolytic protein, plays an important role in both cellulose utilization and cell motility probably by being involved in the appropriate production of outer membrane proteins.

Cell surface properties and biofilm formation of pathogenic bacteria

Cell surface properties of slime production, cell hydrophobicity (CH), colony spreading, auto-aggregation, and biofilm formation (BF) of pathogens were investigated. Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes generally formed strong biofilms and were positive slime producers. The extracellular polymeric substances isolated from agar-grown biofilms of Bacillus cereus and P. aeruginosa contained a high proportion of carbohydrates, proteins, and extracellular DNA. High levels of CH were observed for all pathogens when hydrocarbons were combined with ammonium sulfate (AS). Levels of hydrophobicity based on bacterial adhesion to hydrocarbons using nonane with AS were positively correlated with hydrophobic interaction chromatography assay and contact-angle measurement results. BF and CH values of all pathogens, except E. coli O157:H7 and B. cereus, were highly correlated. Levels of pathogen hydrophobicity differed depending on the measurement method. Factors affecting BF were different depending upon the pathogen.

PilG is Involved in the Regulation of Twitching Motility and Antifungal Antibiotic Biosynthesis in the Biological Control Agent Lysobacter enzymogenes.

Lysobacter enzymogenes strain C3 is a gliding bacterium which produces the antifungal secondary metabolite heat-stable antifungal factor (HSAF) and type IV pilus (T4P) as important mechanisms in biological control activity against fungal pathogens. To date, the regulators that control HSAF biosynthesis and T4P-dependent twitching motility in L. enzymogenes are poorly explored. In the present study, we addressed the role of pilG in the regulation of these two traits in L. enzymogenes. PilG of L. enzymogenes was found to be a response regulator, commonly known as a component of a two-component transduction system. Mutation of pilG in strain C3 abolished its ability to display spreading colony phenotype and cell movement at the colony margin, which is indicative of twitching motility; hence, PilG positively regulates twitching motility in L. enzymogenes. Mutation of pilG also enhanced HSAF production and the transcription of its key biosynthetic gene hsaf pks/nrps, suggesting that PilG plays a negative regulatory role in HSAF biosynthesis. This finding represents the first demonstration of the regulator PilG having a role in secondary metabolite biosynthesis in bacteria. Collectively, our results suggest that key ecological functions (HSAF production and twitching motility) in L. enzymogenes strain C3 are regulated in opposite directions by the same regulatory protein, PilG.

Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus.

ABSTRACTThe nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus.IMPORTANCE We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

A new locus in Cytophaga hutchinsonii involved in colony spreading on agar surfaces and individual cell gliding.

Cytophaga hutchinsonii glides rapidly over surfaces by an unknown mechanism without flagella and type IV pili and it can degrade crystalline cellulose efficiently by a novel mechanism. Tn4351 transposon mutagenesis was used to identify a new gene, CHU_1798, essential for colony spreading on agar surfaces. Further study showed that disruption of CHU_1798 caused non-spreading colonies on both soft and hard agar surfaces and individual cells were partially deficient in gliding on glass surfaces. The CHU_1798 mutant could digest cellulose as long as the cells were in direct contact with the cellulose, but it could not degrade cellulose powder buried in the agar plate. Scanning electron microscopy showed that individual mutant cells arranged irregularly on the cellulose fiber surface at an early stage of incubation, but later showed a regular parallel arrangement when there were plenty of cells and could spread along the cellulose fibers. These results suggest that CHU_1798 plays an important role in the motility of C. hutchinsonii and provide insight into the relation between cell motility and cellulose degradation.

Synthetic vs natural scaffolds for human limbal stem cells.

AimTo investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane.MethodsHuman placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment.ResultsScanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin.ConclusionNatural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purposes.

‘The miserable remnant of this ill-used people’: colonial genocide and the Moriori of New Zealand's Chatham Islands

Genocide scholars have not engaged with the killing of the Moriori people of New Zealand's Chatham Islands by two Māori iwi (tribes). New Zealand historians who have discussed Moriori have not used the paradigms and language of genocide studies. This article argues that Moriori were victims of genocide, and that their experience both challenges and deepens how colonial genocide is understood. Narratives of colonial genocides that assume the destruction of an indigenous people by a colonizing power are inadequate for understanding events on the Chathams, as are genocidal frameworks that assume perpetrators must be state actors. The colonial role was important but indirect: the encounter of Māori with Britain and Australia provided the physical and ideological tools that made the Moriori genocide possible. Māori behaviour on the Chathams diverged from tradition, with the racial tropes, destructive behaviours and justification for invading the Chathams all acquired through the colonial spread of British people into Polynesia. More recently, the use of imported imagery and politicization of the Moriori experience has revealed the consequences of successive generations’ failure to properly remember or even identify the genocide.

Branching instability in expanding bacterial colonies.

Self-organization in developing living organisms relies on the capability of cells to duplicate and perform a collective motion inside the surrounding environment. Chemical and mechanical interactions coordinate such a cooperative behaviour, driving the dynamical evolution of the macroscopic system. In this work, we perform an analytical and computational analysis to study pattern formation during the spreading of an initially circular bacterial colony on a Petri dish. The continuous mathematical model addresses the growth and the chemotactic migration of the living monolayer, together with the diffusion and consumption of nutrients in the agar. The governing equations contain four dimensionless parameters, accounting for the interplay among the chemotactic response, the bacteria-substrate interaction and the experimental geometry. The spreading colony is found to be always linearly unstable to perturbations of the interface, whereas branching instability arises in finite-element numerical simulations. The typical length scales of such fingers, which align in the radial direction and later undergo further branching, are controlled by the size parameters of the problem, whereas the emergence of branching is favoured if the diffusion is dominant on the chemotaxis. The model is able to predict the experimental morphologies, confirming that compact (resp. branched) patterns arise for fast (resp. slow) expanding colonies. Such results, while providing new insights into pattern selection in bacterial colonies, may finally have important applications for designing controlled patterns.

Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion.

Cytophaga hutchinsonii, an aerobic cellulolytic soil bacterium, is capable of degrading crystalline cellulose and gliding over surface rapidly. The involved mechanisms, however, are largely unknown. Here, we used the mariner-based transposon HimarEm1 to screen for C. hutchinsonii mutants deficient in utilizing filter paper as the sole carbon source. A novel gene locus, chu_1719, encoding a hypothetical protein was identified, whose inactivation resulted in a compromised growth of C. hutchinsonii on filter paper. Further analysis revealed that disruption of chu_1719 suppressed colony spreading but had no significant effect on Avicel degradation in liquid medium. Carboxymethylcellulase (CMCase) activity of the mutant membrane proteins was reduced by about 40% as compared with the wild-type strain. Moreover, profiles of cellulose-adsorbed outer membrane proteins were significantly different between the mutant and wild-type (WT) strains. These results suggest that chu_1719 plays an important role in controlling the spreading motility and cellulose utilization probably by affecting the appropriate production of membrane proteins in C. hutchinsonii.

Biomass and root stem production of a colony-forming willow (Salix interior) on highly disturbed, low fertility sites

This study describes biomass production, colony formation, and clonal spread via root stems (RS) of a wide-ranging North American willow species, Salix interior Rowlee (INT), one of the few willows that spread via vegetatively reproduced colonies, which can result in hundreds of upright stems arising from a shallow horizontal root network. Eight INT clones were tested in a common-garden experiment on two distinct site types (shale rock overburden and coarse gravel erosion sediments) with very low nitrogen and nutrients on a former coal mine site. Survival, height growth, aboveground biomass, and number of root stems (NRS) were quantified following 3 years of growth after establishment as rootless stem cuttings. Clonal differences were significant for survival and height at age 3. Survival was significantly greater on the coarse gravel outwash than shale rock overburden after the first and third year, but differences were only significant in the first year. There was a significant positive relationship between height growth and survival, both of which are indicators of plant vigor. Analysis of clonal variation in NRS showed an intermediate level of significance (P = 0.083), and a significant clone by site interaction (P = 0.024). On coarse gravel outwash, the more vigorous clones for height growth also produced more RS, indicating the absence of a potential trade-off in carbohydrate resource allocation between height growth of the ortet (mother plant) and its capacity for colony formation and spread via RS. It appears that RS quickly become independent sources of carbohydrate production. However, there was no relationship between clonal height growth and NRS on the rock overburden. Loose sand and gravel outwash deposits promoted a more rapid spread of the shallow horizontal root network than the less penetrable shale rock overburden that dominates this former coal mine site.

Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence.

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.

The Temporal and Spatial Distribution of Catlinite and Redstone Pipes on Caddo Sites

Catlinite and redstone pipes are widely distributed on post-A.D 1450 native American sites across eastern North America, including the Caddo area of the far Southeast. As Rodning indicates, however, catlinite pipes are much more widespread from the late seventeenth century to the early eighteenth century, where the smoking of catlinite pipes is associated with calumet ceremonialism, and the spread of calumet ceremonialism associated with the “spread of European colonists and colonialism.” In this article, I discuss the temporal and spatial distribution of catlinite and redstone pipes on Caddo sites across the northern and southern Caddo areas. These pipes occur in both pre- and post-European contexts in Caddo sites, and take several forms (disk and elbow pipes), but their widest distribution is on sites that date from ca. A.D. 1690-1780 on Caddo sites in East Texas. Catlinite and redstone pipes are made from argillite found in sources in the Great Plains and the upper Midwest, most notably at Pipestone National Monument in southwestern Minnesota. According to Rodning, the French “colonists are known to have imported argillite and perhaps catlinite from the Midwest or Plains… for the purpose of making calumet pipes to give as gifts to native American groups with whom the French sought alliances.”

Flavobacterium johnsoniae PorV is required for secretion of a subset of proteins targeted to the type IX secretion system.

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.