Involvement of the Cell Division Protein DamX in the Infection Process of Bacteriophage T4.

The molecular mechanism of how the infecting DNA of bacteriophage T4 passes from the capsid through the bacterial cell wall and enters the cytoplasm is essentially unknown. After adsorption, the short tail fibers of the infecting phage extend from the baseplate and trigger the contraction of the tail sheath, leading to a puncturing of the outer membrane by the tail tip needle composed of the proteins gp5.4, gp5 and gp27. To explore the events that occur in the periplasm and at the inner membrane, we constructed T4 phages that have a modified gp27 in their tail tip with a His-tag. Shortly after infection with these phages, cells were chemically cross-linked and solubilized. The cross-linked products were affinity-purified on a nickel column and the co-purified proteins were identified by mass spectrometry, and we found that predominantly the inner membrane proteins DamX, SdhA and PpiD were cross-linked. The same partner proteins were identified when purified gp27 was added to Escherichia coli spheroplasts, suggesting a direct protein-protein interaction.

The Central Spike Complex of Bacteriophage T4 Contacts PpiD in the Periplasm of Escherichia coli.

Infecting bacteriophage T4 uses a contractile tail structure to breach the envelope of the Escherichia coli host cell. During contraction, the tail tube headed with the "central spike complex" is thought to mechanically puncture the outer membrane. We show here that a purified tip fragment of the central spike complex interacts with periplasmic chaperone PpiD, which is anchored to the cytoplasmic membrane. PpiD may be involved in the penetration of the inner membrane by the T4 injection machinery, resulting in a DNA-conducting channel to translocate the phage DNA into the interior of the cell. Host cells with the ppiD gene deleted showed partial reduction in the plating efficiency of T4, suggesting a supporting role of PpiD to improve the efficiency of the infection process.

YidC-mediated membrane insertion.

The most simple membrane protein insertion catalyst known so far is the universal YidC/Oxa/Alb insertase that is composed of a single multi-spanning protein present in archaea, bacteria and in eukaryotic organelles. In bacteria, YidC is known to integrate small membrane proteins on its own and more complex proteins in conjunction with the SecYEG translocase. In mitochondria, the YidC homologue Oxa is responsible for the insertion of all membrane proteins synthesized in the matrix since no Sec homologues are present in the mitochondrial inner membrane. This is tantamount to the observation that YidC is able to operate also independently of SecYEG in bacteria. Reconstituted into liposomes, YidC rapidly and efficiently binds to substrate proteins and leads to their integration into the bilayer. Additionally, single molecule force spectroscopy experiments show that YidC binds to unfolded membrane proteins and promotes their folding into the membrane bilayer. To achieve membrane insertion and the correct folding, the periplasmic regions of the substrate have to cross the membrane with the help of YidC by a mechanism that is presently explored.

Membrane protein insertase YidC in bacteria and archaea.

The insertion of proteins into the prokaryotic plasma membrane is catalyzed by translocases and insertases. On one hand, the Sec translocase operates as a transmembrane channel that can open laterally to first bind and then release the hydrophobic segments of a substrate protein into the lipid bilayer. On the other hand, YidC insertases interact with their substrates in a groove-like structure at an amphiphilic protein-lipid interface thus allowing the transmembrane segments of the substrate to slide into the lipid bilayer. The recently published high-resolution structures of YidC provide new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane.

The C-terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli.

The marine Gram-negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C-terminal regions similar to the YidC homologues in mitochondria and Gram-positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C-terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane. Here, we show in E. coli that an extended C-terminal region of YidC can compensate for a loss of SRP-receptor function in vivo. Furthermore, the enhanced affinity of the ribosome to the chimeric YidC allows the isolation of a ribosome nascent chain complex together with the C-terminally elongated YidC chimera. This complex was visualized at 8.6 Šby cryo-electron microscopy and shows a close contact of the ribosome and a YidC monomer.

The photosensor protein Ppr of Rhodocista centenaria is linked to the chemotaxis signalling pathway.

BACKGROUND: Rhodocista centenaria is a phototrophic α-proteobacterium exhibiting a phototactic behaviour visible as colony movement on agar plates directed to red light. As many phototrophic purple bacteria R. centenaria possesses a soluble photoactive yellow protein (Pyp). It exists as a long fusion protein, designated Ppr, consisting of three domains, the Pyp domain, a putative bilin binding domain (Bbd) and a histidine kinase domain (Pph). The Ppr protein is involved in the regulation of polyketide synthesis but it is still unclear, how this is connected to phototaxis and chemotaxis. RESULTS: To elucidate the possible role of Ppr and Pph in the chemotactic network we studied the interaction with chemotactic proteins in vitro as well as in vivo. Matrix-assisted coelution experiments were performed to study the possible communication of the different putative binding partners. The kinase domain of the Ppr protein was found to interact with the chemotactic linker protein CheW. The formation of this complex was clearly ATP-dependent. Further results indicated that the Pph histidine kinase domain and CheW may form a complex with the chemotactic kinase CheAY suggesting a role of Ppr in the chemotaxis signalling pathway. In addition, when Ppr or Pph were expressed in Escherichia coli, the chemotactic response of the cells was dramatically affected. CONCLUSIONS: The Ppr protein of Rhodocista centenaria directly interacts with the chemotactic protein CheW. This suggests a role of the Ppr protein in the regulation of the chemotactic response in addition to its role in chalcone synthesis.

YidC as an essential and multifunctional component in membrane protein assembly.

Membrane proteins fulfill a number of vital functions in prokaryotic and eukaryotic cells. They are often organized in multicomponent complexes, folded within the membrane bilayer and interacting with the cytoplasmic and periplasmic or external soluble compartments. For the biogenesis of integral membrane proteins, the essential biochemical steps are (1) the insertion and topogenesis of the transmembrane protein segments into the lipid bilayer, (2) the three-dimensional folding of the translocated hydrophilic domains, and (3) the assembly into multimeric complexes. Intensive research has elucidated the basic mechanisms of membrane protein insertion in the homologous translocation machineries of different cellular systems. Whereas the Sec translocation system is found in the endoplasmic reticulum of eukaryotic cells and in the prokaryotic plasma membrane, the YidC-Oxa1 membrane insertase is present in prokaryotic and organellar membranes. This review focuses on the discoveries of the YidC system in bacterial as well as the Oxa1/Alb3 protein family of eukaryotic cells and will particularly emphasize evolutionary aspects.

Different regions of the nonconserved large periplasmic domain of Escherichia coli YidC are involved in the SecF interaction and membrane insertase activity.

The YidC protein of Escherichia coli is required for inserting Sec-independent membrane proteins and has a supportive role for the insertion of Sec-dependent proteins into the membrane bilayer. Because a portion of YidC copurifies with the Sec translocase, this interaction might be necessary to assist in the membrane insertion of Sec-dependent proteins. This study describes a deletion analysis that investigates which parts of YidC are required for its interaction with the SecDF complex of the Sec translocase and for the function of YidC as an insertase for the Sec-dependent membrane proteins. The results suggest that the first periplasmic region, which includes residues 24-346, is required for the interaction of YidC with the Sec translocase, in particular with the SecF protein. Further studies showed that residues 215-265 of YidC are sufficient for SecF binding. Surprisingly, the interaction of YidC with SecF is not critical for cell viability as YidC, lacking residues 24-264, was fully functional to support the growth of E. coli. It was also observed that this YidC mutant was fully functional to insert the Sec-dependent subunit A of the F(1)F(o) ATP synthase and an M13 procoat derivative, as well as the Sec-independent M13 procoat protein and subunit C of the ATP synthase. Only when additional residues of the periplasmic region were deleted (265-346) was the membrane insertase function of YidC inhibited.

The cytoplasmic C-terminal domain of the Escherichia coli KdpD protein functions as a K+ sensor.

The KdpD protein is a K(+) sensor kinase located in the cytoplasmic membrane of Escherichia coli. It contains four transmembrane stretches and two short periplasmic loops of 4 and 10 amino acid residues, respectively. To determine which part of KdpD functions as a K(+) sensor, genetic variants were constructed with truncations or altered arrangements of the transmembrane segments. All KdpD constructs were tested by complementation of an E. coli kdpD deletion strain for their ability to grow at a K(+) concentration of 0.1 mM in the medium. A soluble protein composed of the C-terminal cytoplasmic domain was able to complement the kdpD deletion strain. In addition, analysis of the beta-galactosidase activity of an E. coli strain which carries a transcriptional fusion of the upstream region of the kdpFABC operon and a promoterless lacZ gene revealed that this soluble KdpD mutant responds to changes in the K(+) concentration in the extracellular medium. The results suggest that the sensing and response functions are both located in the C-terminal domain and might be modulated by the N-terminal domain as well as by membrane anchoring.

Escherichia coli YidC is a membrane insertase for Sec-independent proteins.

YidC is a recently discovered bacterial membrane protein that is related to the mitochondrial Oxa1p and the Alb3 protein of chloroplasts. These proteins are required in the membrane integration process of newly synthesized proteins that do not require the classical Sec machinery. Here we demonstrate that YidC is sufficient for the membrane integration of a Sec-independent protein. Microgram amounts of the purified single-spanning Pf3 coat protein were efficiently inserted into proteoliposomes containing the purified YidC. A mutant Pf3 coat protein with an extended hydrophobic region was inserted independently of YidC into the membrane both in vivo and in vitro, but its insertion was accelerated by YidC. These results show that YidC can function separately from the Sec translocase to integrate membrane proteins into the lipid bilayer.