Periplasmic Protease Research Articles

Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor.

The second messenger c-di-GMP (or cyclic diguanylate) regulates biofilm formation, a physiological adaptation process in bacteria, via a widely conserved signaling node comprising a prototypical transmembrane receptor for c-di-GMP, LapD, and a cognate periplasmic protease, LapG. Previously, we reported a structure-function study of a soluble LapD•LapG complex, which established conformational changes in the receptor that lead to c-di-GMP-dependent protease recruitment (Chatterjee et al., 2014). This work also revealed a basal affinity of c-di-GMP-unbound receptor for LapG, the relevance of which remained enigmatic. Here, we elucidate the structural basis of coincidence detection that relies on both c-di-GMP and LapG binding to LapD for receptor activation. The data indicate that high-affinity for LapG relies on the formation of a receptor dimer-of-dimers, rather than a simple conformational change within dimeric LapD. The proposed mechanism provides a rationale of how external proteins can regulate receptor function and may also apply to c-di-GMP-metabolizing enzymes that are akin to LapD.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.

Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells

Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

Molecular characterization of Vibrio harveyi virulence-associated serine protease and outer membrane protein genes for vaccine development

Vibrio harveyi is the most pathogenic species associated with the infection in a wide range of marine species in aquaculture industries. A few virulence-associated genes have been discovered in V. harveyi. This study reports the cloning, sequence analysis and phylogenetic study of serine protease (VHS) and outer membrane protein (OMP) from a pathogenic V. harveyi, which isolated from a local outbreak of diseased tiger grouper. Molecular identification revealed that VHS and OMP consist of 1368 and 816 base pairs and encoded for mature peptides of 429 and 251 amino acids, respectively. The amino acids sequence identities of VHS was 100% similarity with protease of V. harveyi and OMP was 99% of membrane protein of V. harveyi, as compared to published sequence. Phylogenetic analysis and conserved domain search proposed that VHS is a serine endoprotease DegQ and OMP is an OmpK type. Signal peptide, transmembrane beta-barrel and subcellular localization have supported the findings whereby demonstrated VHS belonged to periplasmic serine protease DegQ, composed one beta-barrel and two alpha-barrels region. OMP displayed six beta-barrels and twelve alpha-barrels suggesting it is belonged to outer membrane integral membrane protein, specifically act as a porin type outer membrane protein. Prediction of antigenic sites revealed that VHS composed 62 sites and OMP have 36 antigenic sites, assuming that they can provoke immune response of the infected hosts. In conclusion, it is strongly suggests that both genes can be potentially used for developing an effective live-attenuated vaccine candidate against vibriosis and further be applied in aquaculture industries.

Selective Pressure for Rapid Membrane Integration Constrains the Sequence of Bacterial Outer Membrane Proteins

The Moon-Fleming hydrophobicity scale suggests that the small thermodynamic penalty for inserting a basic residue into the lipid bilayer of the bacterial outer membrane (OM) does not account for the extreme evolutionary selection against arginine residues on the lipid-facing surfaces of OM proteins (OMPs). To explain this phenomenon, we investigated the consequences of introducing lipid-facing arginine residues into the β-barrel of two E. coli OMPs, an autotransporter (EspP) and outer membrane phospholipase A (OmpLA). We found that lipid-facing arginine residues do not affect the targeting of EspP to the Bam complex, an essential hetero-oligomer that catalyzes the membrane integration of OMPs, but markedly slow the membrane integration of the β-barrel domain. OmpLA mutants that contain lipid-facing arginine residues were likewise slowly incorporated into the OM in a strain that lacks the periplasmic protease DegP. In contrast to the EspP variants, however, the OmpLA mutants were degraded before they were inserted into the OM in a wild-type strain. The OmpLA arginine variants also folded slowly into large unilamellar vesicles in vitro. Taken together, the results suggest that lipid-facing arginine residues are strongly disfavored because proteins that assemble slowly are captured by the periplasmic quality control system through a kinetic partitioning mechanism or form prolonged interactions with the Bam complex that are potentially deleterious. Our results not only explain a key constraint on the sequence of OMPs, but also reveal important differences in the early stages of assembly of distinct classes of these proteins.

Controlling the Connections of Cells to the Biofilm Matrix.

The importance of cyclic di-GMP (c-di-GMP) and its control of biofilm matrix assembly and production has been a focal point of researchers in recent history. In this issue, Cooley et al. (Cooley RB, Smith TJ, Leung W, Tierney V, Borlee BR, O'Toole GA, Sondermann H, J Bacteriol 198:66-77, http://dx.doi.org/10.1128/JB.00369-15) demonstrate that two c-di-GMP controlled features of Pseudomonas aeruginosa, the periplasmic protease LapG and the surface adhesin CdrA, are linked. CdrA is shown to be a substrate of LapG, with LapG activity controlled by intracellular c-di-GMP levels. This commentary discusses the significance of this finding.

The LapG protein plays a role in Pseudomonas aeruginosa biofilm formation by controlling the presence of the CdrA adhesin on the cell surface

Pseudomonas aeruginosa is a clinically relevant species involved in biofilm-based chronic infections. We provide evidence that the P. aeruginosa LapG protein functions as a periplasmic protease that can cleave the protein adhesin CdrA off the cell surface, and thereby plays a role in biofilm formation and biofilm dispersal. The P. aeruginosa LapG protein is shown to be a functional homolog of the Pseudomonas putida LapG protein which has previously been shown to function as a periplasmic protease that targets the surface adhesin LapA. Transposon mutagenesis and characterization of defined knockout mutants provided evidence that the CdrA adhesin is a target of LapG in P. aeruginosa. A wspF lapG double mutant was hyper-aggregating and hyper biofilm forming, whereas a wspF lapG cdrA triple mutant lost these phenotypes. In addition, western blot detection of CdrA in culture supernatants and whole-cell protein fractions showed that CdrA was retained in the whole-cell protein fraction when LapG was absent, whereas it was found in the culture supernatant when LapG was present. The finding that CdrA is a target of LapG in P. aeruginosa is surprising because CdrA has no homology to LapA.

Regulated proteolysis of a cross-link–specific peptidoglycan hydrolase contributes to bacterial morphogenesis

Bacterial growth and morphogenesis are intimately coupled to expansion of peptidoglycan (PG), an extensively cross-linked macromolecule that forms a protective mesh-like sacculus around the cytoplasmic membrane. Growth of the PG sacculus is a dynamic event requiring the concerted action of hydrolases that cleave the cross-links for insertion of new material and synthases that catalyze cross-link formation; however, the factors that regulate PG expansion during bacterial growth are poorly understood. Here, we show that the PG hydrolase MepS (formerly Spr), which is specific to cleavage of cross-links during PG expansion in Escherichia coli, is modulated by proteolysis. Using combined genetic, molecular, and biochemical approaches, we demonstrate that MepS is rapidly degraded by a proteolytic system comprising an outer membrane lipoprotein of unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In summary, our results indicate that the NlpI-Prc system contributes to growth and enlargement of the PG sacculus by modulating the cellular levels of the cross-link-cleaving hydrolase MepS. Overall, this study signifies the importance of PG cross-link cleavage and its regulation in bacterial cell wall biogenesis.

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate.

We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger.

ZnO nanoparticles impose a panmetabolic toxic effect along with strong necrosis, inducing activation of the envelope stress response in Salmonella enterica serovar Enteritidis.

In this study, we tested the antimicrobial activity of three metal nanoparticles (NPs), ZnO, MgO, and CaO NPs, against Salmonella enterica serovar Enteritidis in liquid medium and on solid surfaces. Out of the three tested metal NPs, ZnO NPs exhibited the most significant antimicrobial effect both in liquid medium and when embedded on solid surfaces. Therefore, we focused on revealing the mechanisms of surface-associated ZnO biocidal activity. Using the global proteome approach, we report that a great majority (79%) of the altered proteins in biofilms formed by Salmonella enterica serovar Enteritidis were downregulated, whereas a much smaller fraction (21%) of proteins were upregulated. Intriguingly, all downregulated proteins were enzymes involved in a wide range of the central metabolic pathways, including translation; amino acid biosynthetic pathways; nucleobase, nucleoside, and nucleotide biosynthetic processes; ATP synthesis-coupled proton transport; the pentose phosphate shunt; and carboxylic acid metabolic processes, indicating that ZnO NPs exert a panmetabolic toxic effect on this prokaryotic organism. In addition to their panmetabolic toxicity, ZnO NPs induced profound changes in cell envelope morphology, imposing additional necrotic effects and triggering the envelope stress response of Salmonella serovar Enteritidis. The envelope stress response effect activated periplasmic chaperones and proteases, transenvelope complexes, and regulators, thereby facilitating protection of this prokaryotic organism against ZnO NPs.

Steric clashes with bound OMP peptides activate the DegS stress-response protease

Escherichia coli senses envelope stress using a signaling cascade initiated when DegS cleaves a transmembrane inhibitor of a transcriptional activator for response genes. Each subunit of the DegS trimer contains a protease domain and a PDZ domain. During stress, unassembled outer-membrane proteins (OMPs) accumulate in the periplasm and their C-terminal peptides activate DegS by binding to its PDZ domains. In the absence of stress, autoinhibitory interactions, mediated by the L3 loop, stabilize inactive DegS, but it is not known how this autoinhibition is reversed during activation. Here, we show that OMP peptides initiate a steric clash between the PDZ domain and the L3 loop that results in a structural rearrangement of the loop and breaking of autoinhibitory interactions. Many different L3-loop sequences are compatible with activation but those that relieve the steric clash reduce OMP activation dramatically. Our results provide a compelling molecular mechanism for allosteric activation of DegS by OMP-peptide binding.

The Campylobacter jejuni CprRS two-component regulatory system regulates aspects of the cell envelope.

Campylobacter jejuni is a leading cause of food-borne gastroenteritis in humans. It lives commensally in the gastrointestinal tract of animals, and tolerates variable conditions during transit/colonization of susceptible hosts. The C. jejuni CprRS two-component system contains an essential response regulator (CprR), and deletion of the cprS sensor kinase enhances biofilms. We sought to identify CprRS-regulated genes and better understand how the system affects survival. Expression from the cprR promoter was highest during logarithmic growth and dependent on CprS. CprR(D52A) did not support viability, indicating that CprR phosphorylation is essential despite the dispensability of CprS. We identified a GTAAAC consensus bound by the CprR C-terminus; the Asp52 residue of full-length CprR was required for binding, suggesting phosphorylation is required. Transcripts differing in expression in ΔcprS compared with wildtype (WT) contained a putative CprR binding site upstream of their promoter region and encoded htrA (periplasmic protease upstream of cprRS) and peb4 (SurA-like chaperone). Consistent with direct regulation, the CprR consensus in the htrA promoter was bound by CprR(CTD). Finally, ΔhtrA formed enhanced biofilms, and ΔcprS biofilms were suppressed by Mg(2+). CprRS is the first C. jejuni regulatory system shown to control genes related to the cell envelope, the first line of interaction between pathogen and changing environments.

The Inverse Autotransporter Intimin Exports Its Passenger Domain via a Hairpin Intermediate

Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation.

Identifying the Oligomerization State of DegP in the Absence and Presence of Substrate

Bacterial outer membrane proteins (OMPs) are synthesized in the cytoplasm, cross the inner membrane, and are then transported across the periplasm before folding into the outer membrane. Similar pathways are present in both mitochondria and chloroplasts. While unfolded outer membrane proteins (uOMPs) are prone to aggregation, they are able to avoid this off pathway reaction with the help of periplasmic chaperones and proteases. The primary protease that interacts with uOMPs is DegP. DegP is a member of the high temperature requirement A (HTRA) protease family and has been implicated in both the Cpx and σE stress response pathways. DegP sequesters and degrades uOMPS when they accumulate in the periplasm. It has been suggested that DegP functions by transitioning from an inactive hexameric state to an active cage-like oligomeric state of either 12 or 24 subunits when substrate is present. In order to further investigate the relationship between the oligomeric state of DegP and the presence of substrate, we performed sedimentation velocity experiments with and without various uOMPs. This allows us to identify the oligomeric populations of DegP at biological concentrations. Results suggest that DegP has significant populations of multiple oligomeric states even in the absence of substrate. This work contributes to our understanding of OMP biogenesis by identifying the nature of uOMP interactions with an important pathway component.

Activation of the σE-dependent stress pathway by conjugative TraR may anticipate conjugational stress.

Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.

Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2′-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb’s pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.

Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP.

Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling.

Comparative proteomic analysis of the membrane proteins of two Haemophilus parasuis strains to identify proteins that may help in habitat adaptation and pathogenesis.

BackgroundHaemophilus parasuis is the causative agent of Glässer’s disease characterized by polyserositis, arthritis, and meningitis in pig, leading to serious economic loss. Despite many years of study, virulence factors and the mechanisms of the entire infection process remain largely unclear. So two-dimensional gel electrophoresis and mass spectrometry were used to search for distinctions at the membrane protein expression level between two H. parasuis isolates aimed at uncovering some proteins potentially involved in habitat adaption and pathogenesis.ResultsA comparative proteomic approach combining two-dimensional gel electrophoresis with mass spectrometry and tandem mass spectrometry was employed to explore the differences among membrane proteomes of a virulent Haemophilus parasuis strain isolated from the lung of a diseased pig and an avirulent strain isolated from the nasal swab of a healthy pig. Differentially expressed protein spots identified by mass spectrometry were annotated and analyzed by bioinformatic interpretation. The mRNA level was determined by quantitative real-time PCR. Proteins representing diverse functional activities were identified. Among them, the tonB-dependent siderophore receptor was a new discovery highlighted for its activity in iron uptake. In addition, periplasmic serine protease and putrescine/spermidine ABC transporter substrate-binding protein were given focus because of their virulence potential. This study revealed that the differentially expressed proteins were important in either the habitat adaption or pathogenesis of H. parasuis.ConclusionsThe outcome demonstrated the presence of some proteins which raise the speculation for their importance in helping in habitat adaption or pathogenesis within the host.

Antibacterial effect and proteomic analysis of graphene-based silver nanoparticles on a pathogenic bacterium Pseudomonas aeruginosa.

Graphene-based silver nanoparticles (Ag NPs-GE) material has been developed and demonstrated antibacterial effect against Escherichia coli and Pseudomonas aeruginosa. In this study, the antibacterial activity and mechanism on P. aeruginosa were investigated. The experiments results showed the minimum bactericidal concentration of Ag NPs-GE to P. aeruginosa is 20μg/ml. When P. aeruginosa were exposed to 20μg/ml Ag NPs-GE for 1h, the cell wall was breakdown. In order to study the mechanism of antibacterial effect of Ag NPs-GE, two-dimensional electrophoresis was carried out to compare the protein expressional profiles of P. aeruginosa exposed to 5μg/ml Ag NPs-GE or 5μg/ml AgNO3 with the untreated bacteria. Identification of differentially expressed protein was performed by MALDI-TOF/TOF MS. The change of proteomic profile induced by Ag NPs-GE was distinct from that induced by AgNO3. Seven identified proteins were found induced and nine proteins were suppressed by Ag NPs-GE. Five identified proteins were found induced and twenty proteins were suppressed by AgNO3. In addition, either Ag NPs-GE or AgNO3 suppressed the expression of eight proteins, amidotransferase, 30S ribosomal protein S6, bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase, arginyl-tRNA synthetase, nitroreductase, acetolactate synthase 3, methionyl-tRNA synthetase and periplasmic tail-specific protease. Furthermore, gene ontology analysis and KEGG pathway analysis were used to characterize the functions of those proteins.

Structural features of the Pseudomonas fluorescens biofilm adhesin LapA required for LapG-dependent cleavage, biofilm formation, and cell surface localization.

The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins.