Regulons and protein-protein interactions of PRD-containing Bacillus anthracis virulence regulators reveal overlapping but distinct functions.

Bacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed 'PCVRs' (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA level or activity result in capBCADE transcription in strains lacking acpA and acpB. We used RNA-Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Plasmid and chromosome-borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥ 4-fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. We determined that AcpA and AcpB form homomultimers, as shown previously for AtxA, and we detected AtxA-AcpA heteromultimers. In co-expression experiments, AcpA activity was reduced by increased levels of AtxA. Our data show that the PCVRs have specific and overlapping activity and that PCVR stoichiometry and potential heteromultimerization can influence target gene expression.

Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance.

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

The fibronectin-binding protein EfbA contributes to pathogenesis and protects against infective endocarditis caused by Enterococcus faecalis.

EfbA is a PavA-like fibronectin adhesin of Enterococcus faecalis previously shown to be important in experimental urinary tract infection. Here, we expressed and purified the E. faecalis OG1RF EfbA and confirmed that this protein binds with high affinity to immobilized fibronectin, collagen I, and collagen V. We constructed an efbA deletion mutant and demonstrated that its virulence was significantly attenuated (P < 0.0006) versus the wild type in a mixed inoculum rat endocarditis model. Furthermore, efbA deletion resulted in diminished ability to bind fibronectin (P < 0.0001) and reduced biofilm (P < 0.001). Reintroduction of efbA into the original chromosomal location restored virulence, adherence to fibronectin, and biofilm formation to wild-type levels. Finally, vaccination of rats with purified recombinant EfbA protein protected against OG1RF endocarditis (P = 0.008 versus control). Taken together, our results demonstrate that EfbA is an important factor involved in E. faecalis endocarditis and that rEfbA immunization is effective in preventing such infection, likely by interfering with bacterial adherence.

The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT.

UNLABELLED: The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of ß-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. IMPORTANCE: Enterococcus faecalis is among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles in E. faecalis pathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA in E. faecalis. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability of E. faecalis to cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.

The two-component system GrvRS (EtaRS) regulates ace expression in Enterococcus faecalis OG1RF.

Expression of ace (adhesin to collagen of Enterococcus faecalis), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46 °C. However, the mechanism of ace/Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator of ace expression under these stress conditions. Using Northern hybridization and ß-galactosidase assays of an ace promoter-lacZ fusion, we found transcription of ace to be virtually absent in a grvR deletion mutant under the conditions that increase ace expression in wild-type OG1RF and in the complemented strain. Moreover, a grvR mutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control of ace expression and E. faecalis virulence.

Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R.

During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs.

Enterococcus faecalis reconfigures its transcriptional regulatory network activation at different copper levels.

A global transcriptional regulatory network was generated in the pathogenic bacterium Enterococcus faecalis in order to understand how this organism can activate and coordinate its expression at different copper concentrations. The topological evaluation of the network showed common patterns described in other organisms. Integrating microarray experiments allowed the identification of two sub-networks activated at low (0.05 mM CuSO4) and high (0.5 mM CuSO4) concentrations of copper. The analysis indicates the presence of specific functionally activated modules induced by copper levels, highlighting the regulons LysR and ArgR as global regulators and CopY, Fur and LexA as local regulators. Taking advantage of the fact that E. faecalis presented a homeostatic module, we produced an in vivo intervention by removing this system from the cell without affecting the connectivity of the global transcriptional network. This strategy led us to find that this bacterium can reconfigure its gene expression to maintain cellular homeostasis, activating new modules principally related to glucose metabolism and transcriptional processes. Finally, these results position E. faecalis as the most complete and controllable systemic model organism for copper homeostasis available to date.

Expression of the collagen adhesin ace by Enterococcus faecalis strain OG1RF is not repressed by Ers but requires the Ers box.

Expression of adhesin to collagen of Enterococcus faecalis (ace), a known virulence factor, is increased by environmental signals such as the presence of serum, high temperature, and bile salts. Currently, the enterococcal regulator of survival (Ers) of E. faecalis strain JH2-2 is the only reported repressor of ace. Here, we show that for strain OG1RF, Ers is not involved in the regulation of ace. Our data showed similar levels of ace expression by OG1RF and its Δers derivative in the presence of bile salts, serum, and high temperature. Using ace promoter-lacZ fusions and site-directed mutagenesis, we confirmed these results and further showed that, while the previously designated Ers box is important for increased expression from the ace promoter of OG1RF, the region responsible for the increase is bigger than the Ers box. In summary, these results indicate that, in strain OG1RF, Ers is not a repressor of ace expression. Although JH2-2 and OG1RF differ by six nucleotides in the region upstream of ace as well as in production of Fsr and gelatinase, the reason(s) for the difference in ace expression between JH2-2 and OG1RF and for increased ace expression in bile, serum and at 46 °C remain(s) to be determined.

Transcriptome response to nitrosative stress in Rhodobacter sphaeroides 2.4.1.

The facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1 has a nitric oxide-response transcriptional regulator, NnrR, and nitric oxide reductase (NOR), although it is incapable of denitrification. To investigate at the genomic level the physiological response to nitrosative stress of R. sphaeroides, the transcriptome profiles of strain 2.4.1 and its NnrR mutant were analyzed before and after exposure to nitrosating agents, S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), under microaerobic conditions. GSNO and SNP affected the expression of different but overlapping sets of genes. Only a limited number of these genes, including the genes for NOR, were under the control of NnrR, and those genes were significantly upregulated by GSNO and by SNP. The oxygen-responsive regulator FnrL and a predicted iron-sensing regulator were perhaps also involved in the transcriptome response to reactive nitrogen species. Some genes, including hemN for heme biosynthesis, were subject to dual regulation by NnrR and FnrL.

Complete genome sequence of Enterococcus faecium strain TX16 and comparative genomic analysis of Enterococcus faecium genomes.

BACKGROUND: Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references. RESULTS: In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3-4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported. CONCLUSIONS: Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined.

Genomic and SNP analyses demonstrate a distant separation of the hospital and community-associated clades of Enterococcus faecium.

Recent studies have pointed to the existence of two subpopulations of Enterococcus faecium, one containing primarily commensal/community-associated (CA) strains and one that contains most clinical or hospital-associated (HA) strains, including those classified by multi-locus sequence typing (MLST) as belonging to the CC17 group. The HA subpopulation more frequently has IS16, pathogenicity island(s), and plasmids or genes associated with antibiotic resistance, colonization, and/or virulence. Supporting the two clades concept, we previously found a 3-10% difference between four genes from HA-clade strains vs. CA-clade strains, including 5% difference between pbp5-R of ampicillin-resistant, HA strains and pbp5-S of ampicillin-sensitive, CA strains. To further investigate the core genome of these subpopulations, we studied 100 genes from 21 E. faecium genome sequences; our analyses of concatenated sequences, SNPs, and individual genes all identified two distinct groups. With the concatenated sequence, HA-clade strains differed by 0-1% from one another while CA clade strains differed from each other by 0-1.1%, with 3.5-4.2% difference between the two clades. While many strains had a few genes that grouped in one clade with most of their genes in the other clade, one strain had 28% of its genes in the CA clade and 72% in the HA clade, consistent with the predicted role of recombination in the evolution of E. faecium. Using estimates for Escherichia coli, molecular clock calculations using sSNP analysis indicate that these two clades may have diverged ≥1 million years ago or, using the higher mutation rate for Bacillus anthracis, ∼300,000 years ago. These data confirm the existence of two clades of E. faecium and show that the differences between the HA and CA clades occur at the core genomic level and long preceded the modern antibiotic era.

Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO(2) -signalling.

AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis. The 475 amino acid sequence of AtxA reveals DNA binding motifs and regions similar to proteins associated with the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope-tagged AtxA proteins to examine protein-protein interactions in cell lysates and in solutions of purified protein. Co-affinity purification, non-denaturing polyacrylamide gel electrophoresis and bis(maleimido)hexane (BMH) cross-linking experiments revealed AtxA homo-multimers. Dimers were the most abundant species. BMH cross-links available cysteines within 13 Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross-linking. All mutants multimerized, but one mutation, C402S, prevented cross-linking. Thus, BMH uses C402 to make the inter-molecular bond between AtxA proteins, but C402 is not required for protein-protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Finally, cultures grown with elevated CO(2) /bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO(2) /bicarbonate, suggesting that this host-associated signal enhances AtxA function by shifting the dimer/monomer equilibrium towards the dimeric state.

Adaptation of aerobic respiration to low O2 environments.

Aerobic respiration in bacteria, Archaea, and mitochondria is performed by oxygen reductase members of the heme-copper oxidoreductase superfamily. These enzymes are redox-driven proton pumps which conserve part of the free energy released from oxygen reduction to generate a proton motive force. The oxygen reductases can be divided into three main families based on evolutionary and structural analyses (A-, B- and C-families), with the B- and C-families evolving after the A-family. The A-family utilizes two proton input channels to transfer protons for pumping and chemistry, whereas the B- and C-families require only one. Generally, the B- and C-families also have higher apparent oxygen affinities than the A-family. Here we use whole cell proton pumping measurements to demonstrate differential proton pumping efficiencies between representatives of the A-, B-, and C-oxygen reductase families. The A-family has a coupling stoichiometry of 1 H(+)/e(-), whereas the B- and C-families have coupling stoichiometries of 0.5 H(+)/e(-). The differential proton pumping stoichiometries, along with differences in the structures of the proton-conducting channels, place critical constraints on models of the mechanism of proton pumping. Most significantly, it is proposed that the adaptation of aerobic respiration to low oxygen environments resulted in a concomitant reduction in energy conservation efficiency, with important physiological and ecological consequences.

The use of chromatin immunoprecipitation to define PpsR binding activity in Rhodobacter sphaeroides 2.4.1.

The expression of genes involved in photosystem development in Rhodobacter sphaeroides is dependent upon three major regulatory networks: FnrL, the PrrBA (RegBA) two-component system, and the transcriptional repressor/antirepressor PpsR/AppA. Of the three regulators, PpsR appears to have the narrowest range of physiological effects, which are limited to effects on the structural and pigment biosynthetic activities involved in photosynthetic membrane function. Although a PrrA(-) mutant is unable to grow under photosynthetic conditions, when a ppsR mutation was present, photosynthetic growth occurred. An examination of the double mutant under anaerobic-dark-dimethyl sulfoxide conditions using microarray analysis revealed the existence of an "extended" PpsR regulon and new physiological roles. To characterize the PpsR regulon and to better ascertain the significance of degeneracy within the PpsR binding sequence in vivo, we adapted the chromatin immunoprecipitation technique to R. sphaeroides. We demonstrated that in vivo there was direct and significant binding by PpsR to newly identified genes involved in microaerobic respiration and periplasmic stress resistance, as well as to photosynthesis genes. The new members of the PpsR regulon are located outside the photosynthesis gene cluster and have degenerate PpsR binding sequences. The possible interaction under physiologic conditions with degenerate binding sequences in the presence of other biologically relevant molecules is discussed with respect to its importance in physiological processes and to the existence of complex phenotypes associated with regulatory mutants. This study further defines the DNA structure necessary for PpsR binding in situ.

Role of the global transcriptional regulator PrrA in Rhodobacter sphaeroides 2.4.1: combined transcriptome and proteome analysis.

The PrrBA two-component regulatory system is a major global regulator in Rhodobacter sphaeroides 2.4.1. Here we have compared the transcriptome and proteome profiles of the wild-type (WT) and mutant PrrA2 cells grown anaerobically in the dark with dimethyl sulfoxide as an electron acceptor. Approximately 25% of the genes present in the PrrA2 genome are regulated by PrrA at the transcriptional level, either directly or indirectly, by twofold or more relative to the WT. The genes affected are widespread throughout all COG (cluster of orthologous group) functional categories, with previously unsuspected "metabolic" genes affected in PrrA2 cells. PrrA was found to act as both an activator and a repressor of transcription, with more genes being repressed in the presence of PrrA (9:5 ratio). An analysis of the genes encoding the 1,536 peptides detected through our chromatographic study, which corresponds to 36% coverage of the genome, revealed that approximately 20% of the genes encoding these proteins were positively regulated, whereas approximately 32% were negatively regulated by PrrA, which is in excellent agreement with the percentages obtained for the whole-genome transcriptome profile. In addition, comparison of the transcriptome and proteome mean parameter values for WT and PrrA2 cells showed good qualitative agreement, indicating that transcript regulation paralleled the corresponding protein abundance, although not one for one. The microarray analysis was validated by direct mRNA measurement of randomly selected genes that were both positively and negatively regulated. lacZ transcriptional and kan translational fusions enabled us to map putative PrrA binding sites and revealed potential gene targets for indirect regulation by PrrA.

A computational strategy to analyze label-free temporal bottom-up proteomics data.

Biological systems are in a continual state of flux, which necessitates an understanding of the dynamic nature of protein abundances. The study of protein abundance dynamics has become feasible with recent improvements in mass spectrometry-based quantitative proteomics. However, a number of challenges still remain related to how best to extract biological information from dynamic proteomics data, for example, challenges related to extraneous variability, missing abundance values, and the identification of significant temporal patterns. This paper describes a strategy that addresses these issues and demonstrates its values for analyzing temporal bottom-up proteomics data using data from a Rhodobacter sphaeroides 2.4.1 time-course study.

Transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration in Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the aa3 cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration. Analyses of these data make it clear that numerous regulatory activities come into play during the transition from one homeostatic state to another.

Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins.

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc(1) complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.

Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases.

Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb3 oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb3 oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm.

Postgenomic adventures with Rhodobacter sphaeroides.

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.