Mechanistic Insight into TrimethylamineN-Oxide Recognition by the Marine Bacterium Ruegeria pomeroyi DSS-3

ABSTRACTTrimethylamineN-oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria.Ruegeria pomeroyiDSS-3, a marineRoseobacter, has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX ofR. pomeroyiDSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca2+ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between “open” and “closed” states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marineRoseobacterclade. This study sheds light on how marine microorganisms utilize TMAO.IMPORTANCETrimethylamineN-oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX ofRuegeria pomeroyiDSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling.

Download Full-text

Molecular Basis of Unexpected Specificity of ABC Transporter-Associated Substrate-Binding Protein DppA from Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.00400-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 1

Author(s):

Mohammad M. Rahman ◽

Mayra A. Machuca ◽

Mohammad F. Khan ◽

Christopher K. Barlow ◽

Ralf B. Schittenhelm ◽

...

Keyword(s):

Helicobacter Pylori ◽

Abc Transporter ◽

Binding Protein ◽

Substrate Binding ◽

Binding Pocket ◽

Amino Acid Sequences ◽

Content Type ◽

H Pylori ◽

Substrate Binding Protein ◽

Broad Specificity

ABSTRACT The gastric pathogen Helicobacter pylori has limited ability to use carbohydrates as a carbon source, relying instead on exogenous amino acids and peptides. Uptake of certain peptides by H. pylori requires an ATP binding cassette (ABC) transporter annotated dipeptide permease (Dpp). The transporter specificity is determined by its cognate substrate-binding protein DppA, which captures ligands in the periplasm and delivers them to the permease. Here, we show that, unlike previously characterized DppA proteins, H. pylori DppA binds, with micromolar affinity, peptides of diverse amino acid sequences ranging between two and eight residues in length. We present analysis of the 1.45-Å-resolution crystal structure of its complex with the tetrapeptide STSA, which provides a structural rationale for the observed broad specificity. Analysis of the molecular surface revealed a ligand-binding pocket that is large enough to accommodate peptides of up to nine residues in length. The structure suggests that H. pylori DppA is able to recognize a wide range of peptide sequences by forming interactions primarily with the peptide main chain atoms. The loop that terminates the peptide-binding pocket in DppAs from other bacteria is significantly shorter in the H. pylori protein, providing an explanation for its ability to bind longer peptides. The subsites accommodating the two N-terminal residues of the peptide ligand make the greatest contribution to the protein-ligand binding energy, in agreement with the observation that dipeptides bind with affinity close to that of longer peptides. IMPORTANCE The World Health Organization listed Helicobacter pylori as a high-priority pathogen for antibiotic development. The potential of using peptide transporters in drug design is well recognized. We discovered that the substrate-binding protein of the ABC transporter for peptides, termed dipeptide permease, is an unusual member of its family in that it directly binds peptides of diverse amino acid sequences, ranging between two and eight residues in length. We also provided a structural rationale for the observed broad specificity. Since the ability to import peptides as a source of carbon is critical for H. pylori, our findings will inform drug design strategies based on inhibition or fusion of membrane-impermeant antimicrobials with peptides.

Download Full-text

Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family

Infection and Immunity ◽

10.1128/iai.00962-19 ◽

2020 ◽

Vol 88 (4) ◽

Author(s):

Julia Cuellar ◽

Mia Åstrand ◽

Heli Elovaara ◽

Annukka Pietikäinen ◽

Saija Sirén ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Cellular Localization ◽

De Novo ◽

Binding Protein ◽

Substrate Binding ◽

Nucleoside Transporter ◽

Bacterial Survival ◽

Content Type ◽

Transporter Family ◽

Substrate Binding Protein

ABSTRACT Borrelia burgdorferi sensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato. Because B. burgdorferi sensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

Download Full-text

Transcriptional Control in Marine Copiotrophic and Oligotrophic Bacteria with Streamlined Genomes

Applied and Environmental Microbiology ◽

10.1128/aem.01299-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 6010-6018 ◽

Cited By ~ 17

Author(s):

Matthew T. Cottrell ◽

David L. Kirchman

Keyword(s):

Marine Bacteria ◽

Environmental Conditions ◽

Transcriptional Control ◽

Heterotrophic Bacteria ◽

Content Type ◽

Oligotrophic Bacteria ◽

Protein Encoding ◽

Culture Growth ◽

Ruegeria Pomeroyi ◽

And Shifts

ABSTRACTBacteria often respond to environmental stimuli using transcriptional control, but this may not be the case for marine bacteria such as “CandidatusPelagibacter ubique,” a cultivated representative of the SAR11 clade, the most abundant organism in the ocean. This bacterium has a small, streamlined genome and an unusually low number of transcriptional regulators, suggesting that transcriptional control is low inPelagibacterand limits its response to environmental conditions. Transcriptome sequencing during batch culture growth revealed that only 0.1% of protein-encoding genes appear to be under transcriptional control inPelagibacterand in another oligotroph (SAR92) whereas >10% of genes were under transcriptional control in the copiotrophsPolaribactersp. strain MED152 andRuegeria pomeroyi. When growth levels changed, transcript levels remained steady inPelagibacterand SAR92 but shifted in MED152 andR. pomeroyi. Transcript abundances per cell, determined using an internal RNA sequencing standard, were low (<1 transcript per cell) for all but a few of the most highly transcribed genes in all four taxa, and there was no correlation between transcript abundances per cell and shifts in the levels of transcription. These results suggest that low transcriptional control contributes to the success ofPelagibacterand possibly other oligotrophic microbes that dominate microbial communities in the oceans.IMPORTANCEDiverse heterotrophic bacteria drive biogeochemical cycling in the ocean. The most abundant types of marine bacteria are oligotrophs with small, streamlined genomes. The metabolic controls that regulate the response of oligotrophic bacteria to environmental conditions remain unclear. Our results reveal that transcriptional control is lower in marine oligotrophic bacteria than in marine copiotrophic bacteria. Although responses of bacteria to environmental conditions are commonly regulated at the level of transcription, metabolism in the most abundant bacteria in the ocean appears to be regulated by other mechanisms.

Download Full-text

Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.00687-11 ◽

2011 ◽

Vol 77 (16) ◽

pp. 5730-5738 ◽

Cited By ~ 36

Author(s):

Hanna M. Dudek ◽

Gonzalo de Gonzalo ◽

Daniel E. Torres Pazmiño ◽

Piotr Stępniak ◽

Lucjan S. Wyrwicz ◽

...

Keyword(s):

Substrate Specificity ◽

Binding Site ◽

Active Site ◽

Structural Model ◽

Mutational Analysis ◽

Substrate Binding ◽

Thermobifida Fusca ◽

Content Type ◽

Substrate Binding Site

ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

Download Full-text

Structural Insights into the Multispecific Recognition of Dipeptides of Deep-Sea Gram-Negative Bacterium Pseudoalteromonas sp. Strain SM9913

Journal of Bacteriology ◽

10.1128/jb.02600-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1125-1134 ◽

Cited By ~ 5

Author(s):

Chun-Yang Li ◽

Xiu-Lan Chen ◽

Qi-Long Qin ◽

Peng Wang ◽

Wei-Xin Zhang ◽

...

Keyword(s):

Nitrogen Cycling ◽

Marine Bacteria ◽

Deep Sea ◽

Substrate Binding ◽

Substrate Recognition ◽

Recognition Mechanism ◽

Gram Negative ◽

Content Type ◽

Peptide Uptake ◽

Structural Insights

ABSTRACTPeptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease (Dpp; an important peptide transporter in bacteria) and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-seaPseudoalteromonassp. strain SM9913 DppA (PsDppA) was analyzed for 25 different dipeptides with various properties by using isothermal titration calorimetry measurements.PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism ofPsDppA, we solved the crystal structures of unligandedPsDppA and ofPsDppA in complex with 4 different types of dipeptides (Ala-Phe, Met-Leu, Gly-Glu, and Val-Thr).PsDppA alternates between an “open” and a “closed” form during substrate binding. Structural analyses of the 4PsDppA-substrate complexes combined with mutational assays indicate thatPsDppA binds to different substrates through a precise mechanism: dipeptides are bound mainly by the interactions between their backbones andPsDppA, in particular by anchoring their N and C termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; and Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism ofPsDppA may be common in Gram-negative bacteria. All together, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake.IMPORTANCEPeptide uptake plays a significant role in nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria recognize and absorb peptides is still unclear. This study analyzed the substrate binding specificity of deep-seaPseudoalteromonassp. strain SM9913 DppA (PsDppA; the dipeptide-binding protein of dipeptide permease) and solved the crystal structures of unligandedPsDppA andPsDppA in complex with 4 different types of dipeptides. The multispecific recognition mechanism ofPsDppA for dipeptides is explained based on structural and mutational analyses. We also find that the substrate-binding mechanism ofPsDppA may be common in Gram-negative bacteria. This study sheds light on marine Gram-negative bacterial peptide uptake and marine nitrogen cycling.

Download Full-text

Structural insights into the substrate specificity of SP_0149, the substrate-binding protein of a methionine ABC transporter from Streptococcus pneumoniae

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19009038 ◽

2019 ◽

Vol 75 (7) ◽

pp. 520-528

Author(s):

Bhavya Jha ◽

Rajan Vyas ◽

Jaya Bhushan ◽

Devinder Sehgal ◽

Bichitra Kumar Biswal

Keyword(s):

Streptococcus Pneumoniae ◽

Substrate Specificity ◽

Rational Design ◽

Binding Protein ◽

Substrate Binding ◽

Three Dimensional ◽

Cumulative Effect ◽

Opportunistic Pathogen ◽

Dimensional Structure ◽

Substrate Binding Protein

Successful pathogenesis is a cumulative effect of the virulence factors of a pathogen and its capability to efficiently utilize the available nutrients from the host. Streptococcus pneumoniae, a Gram-positive opportunistic pathogen, may either reside asymptomatically as a nasopharyngeal commensal inside the human host or cause lethal diseases, including pneumonia, meningitis and sepsis. S. pneumoniae is known to acquire methionine (Met) from its host through a Met importer. Here, the crystal structure of the substrate-binding protein (SBP; SP_0149) of an ABC importer with Met bound is reported at a resolution of 1.95 Å. The three-dimensional structure of SBP shows that it is composed of two distinct domains, each consisting of a mixed β-sheet flanked by helices. The substrate, Met, is bound in the central part of the interface between the two domains. The overall structure of SP_0149 resembles those of SBPs from other reported bacterial Met and Gly-Met dipeptide transporters. However, a detailed analysis of these structures shows notable variations in the amino-acid composition of the substrate-binding pockets of the SP_0149–Met and GmpC–Gly-Met structures. In particular, SP_0149 harbors Thr212 and Tyr114, whereas the corresponding residues in GmpC are Gly and Val. This difference is likely to be the underlying basis for their differential substrate specificity. In summary, the structure of the SP_0149–Met complex provides insights into the transport function of SP_0149 and its interactions with methionine. It opens up avenues for the rational design of inhibitors of SP_0149 through a structure-mediated approach.

Download Full-text

OpuF, a NewBacillusCompatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain

Applied and Environmental Microbiology ◽

10.1128/aem.01728-18 ◽

2018 ◽

Vol 84 (20) ◽

Cited By ~ 7

Author(s):

Laura Teichmann ◽

Henriette Kümmel ◽

Bianca Warmbold ◽

Erhard Bremer

Keyword(s):

Abc Transporters ◽

Transmembrane Domain ◽

Binding Protein ◽

Substrate Binding ◽

Compatible Solutes ◽

Compatible Solute ◽

Content Type ◽

High Osmolarity ◽

Physiological Characterization ◽

Substrate Binding Protein

ABSTRACTThe accumulation of compatible solutes is a common defense of bacteria against the detrimental effects of high osmolarity. Uptake systems for these compounds are cornerstones in cellular osmostress responses because they allow the energy-preserving scavenging of osmostress protectants from environmental sources.Bacillus subtilisis well studied with respect to the import of compatible solutes and its five transport systems (OpuA, OpuB, OpuC, OpuD, and OpuE), for these stress protectants have previously been comprehensively studied. Building on this knowledge and taking advantage of the unabated appearance of new genome sequences of members of the genusBacillus, we report here the discovery, physiological characterization, and phylogenomics of a new member of the Opu family of transporters, OpuF (OpuFA-OpuFB). OpuF is not present inB. subtilisbut it is widely distributed in members of the large genusBacillus. OpuF is a representative of a subgroup of ATP-binding cassette (ABC) transporters in which the substrate-binding protein (SBP) is fused to the transmembrane domain (TMD). We studied the salient features of the OpuF transporters fromBacillus infantisandBacillus panaciterraeby functional reconstitution in aB. subtilischassis strain lacking known Opu transporters. A common property of the examined OpuF systems is their substrate profile; OpuF mediates the import of glycine betaine, proline betaine, homobetaine, and the marine osmolyte dimethylsulfoniopropionate (DMSP). Anin silicomodel of the SBP domain of the TMD-SBP hybrid protein OpuFB was established. It revealed the presence of an aromatic cage, a structural feature commonly present in ligand-binding sites of compatible solute importers.IMPORTANCEThe high-affinity import of compatible solutes from environmental sources is an important aspect of the cellular defense of many bacteria and archaea against the harmful effects of high external osmolarity. The accumulation of these osmostress protectants counteracts high-osmolarity-instigated water efflux, a drop in turgor to nonphysiological values, and an undue increase in molecular crowding of the cytoplasm; they thereby foster microbial growth under osmotically unfavorable conditions. Importers for compatible solutes allow the energy-preserving scavenging of osmoprotective and physiologically compliant organic solutes from environmental sources. We report here the discovery, exemplary physiological characterization, and phylogenomics of a new compatible solute importer, OpuF, widely found in members of theBacillusgenus. The OpuF system is a representative of a growing subgroup of ABC transporters in which the substrate-scavenging function of the substrate-binding protein (SBP) and the membrane-embedded substrate translocating subunit (TMD) are fused into a single polypeptide chain.

Download Full-text

Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis

Infection and Immunity ◽

10.1128/iai.01832-14 ◽

2014 ◽

Vol 82 (8) ◽

pp. 3503-3512 ◽

Cited By ~ 13

Author(s):

Taketo Otsuka ◽

Charmaine Kirkham ◽

Antoinette Johnson ◽

Megan M. Jones ◽

Timothy F. Murphy

Keyword(s):

Otitis Media ◽

Moraxella Catarrhalis ◽

Binding Protein ◽

Substrate Binding ◽

Vaccine Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Chronic Obstructive ◽

Content Type ◽

Substrate Binding Protein

ABSTRACTMoraxella catarrhalisis a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeableHaemophilus influenzae,M. catarrhalishas become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, ofM. catarrhalis. Among 30 clinical isolates tested, thesbp2gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not thesbp2mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance ofM. catarrhalisfrom the lung compared to that in the control group at both 25-μg and 50-μg doses (P< 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen againstM. catarrhalis.

Download Full-text

Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

Infection and Immunity ◽

10.1128/iai.02185-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4758-4766 ◽

Cited By ~ 29

Author(s):

Megan M. Jones ◽

Antoinette Johnson ◽

Mary Koszelak-Rosenblum ◽

Charmaine Kirkham ◽

Aimee L. Brauer ◽

...

Keyword(s):

Amino Acid ◽

Respiratory Tract ◽

Moraxella Catarrhalis ◽

Minimal Medium ◽

Substrate Binding ◽

Amino Acid Residues ◽

Wild Type ◽

Content Type ◽

Oligopeptide Permease ◽

Substrate Binding Protein

ABSTRACTMoraxella catarrhalisis a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality.M. catarrhalishas a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival.M. catarrhalishas a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB,oppC,oppD,oppF, andoppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which theoppBCDFAgene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth ofM. catarrhalisis important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-typeM. catarrhaliswas observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requiresl-arginine. AnoppAknockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.

Download Full-text

The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis

Infection and Immunity ◽

10.1128/iai.00799-15 ◽

2015 ◽

Vol 84 (2) ◽

pp. 432-438 ◽

Cited By ~ 10

Author(s):

Taketo Otsuka ◽

Charmaine Kirkham ◽

Aimee Brauer ◽

Mary Koszelak-Rosenblum ◽

Michael G. Malkowski ◽

...

Keyword(s):

Amino Acids ◽

Respiratory Tract ◽

Abc Transporter ◽

Moraxella Catarrhalis ◽

Vaccine Candidate ◽

Binding Protein ◽

Substrate Binding ◽

Content Type ◽

Basic Amino Acids ◽

Substrate Binding Protein

Moraxella catarrhalisis an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to preventM. catarrhalisinfections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface ofM. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement ofM. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth ofM. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen.

Download Full-text