The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.

The ß-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, ß-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli.

The reducible complexity of a mitochondrial molecular machine.

Molecular machines drive essential biological processes, with the component parts of these machines each contributing a partial function or structural element. Mitochondria are organelles of eukaryotic cells, and depend for their biogenesis on a set of molecular machines for protein transport. How these molecular machines evolved is a fundamental question. Mitochondria were derived from an alpha-proteobacterial endosymbiont, and we identified in alpha-proteobacteria the component parts of a mitochondrial protein transport machine. In bacteria, the components are found in the inner membrane, topologically equivalent to the mitochondrial proteins. Although the bacterial proteins function in simple assemblies, relatively little mutation would be required to convert them to function as a protein transport machine. This analysis of protein transport provides a blueprint for the evolution of cellular machinery in general.

Evidence of a reduced and modified mitochondrial protein import apparatus in microsporidian mitosomes.

Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.

A mortise-tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes.

Bacterial autotransporters comprise a 12-stranded membrane-embedded ß-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring ß-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

A bioinformatic strategy for the detection, classification and analysis of bacterial autotransporters.

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.

Discovery of an archetypal protein transport system in bacterial outer membranes.

Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

Using hidden markov models to discover new protein transport machines.

Protein import and export pathways are driven by protein translocases, often comprised of multiple subunits, and usually conserved across a range of organisms. Protein import into mitochondria is fundamental to eukaryotic organisms and is initiated when substrate proteins are translocated across the mitochondrial outer membrane by the TOM complex. The essential subunit of this complex is a protein called Tom40, which is probably a beta-barrel in structure and serves as the translocation pore. We describe a hidden Markov model search designed to find the Tom40 sequence in the amoeba Entamoeba histolytica. This organism has a highly reduced "mitosome", an organelle whose relationship to mitochondria has been the subject of controversy. The Tom40 sequence could not be found with BLAST-based searches, but a hidden Markov model search identified a likely candidate to form the protein import pore in the outer mitosomal membrane in E. histolytica.