1. Bacterial Cell Wall Growth, Shape and Division

Adeline Derouaux, Mohammed Terrak, Tanneke den Blaauwen and Waldemar Vollmer

The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by outer-membrane regulators of peptidoglycan synthases. Cell division is achieved by a large assembly of essential cell division proteins, the divisome, that coordinates the synthesis and hydrolysis of peptidoglycan during septation. Coccal species such as Staphylococcus aureus grow exclusively by synthesis and cleavage of a cross-wall. Ovococci like Streptococcus pneumoniae elongate at a central growth zone resulting in their lancet-shape. Rod-shaped species elongate either at the side-wall coordinated by the MreB cytoskeleton, like Escherichia coli or Bacillus subtilis, or at the poles like Corynebacterium glutamicum. Bacteria have different mechanisms to achieve bent or helical cell shape, involving cytoskeletal proteins, periplasmic flagella or peptidoglycan hydrolases, and to form branched, filamentous cell chains. Peptidoglycan enzymes and cytoskeletal proteins are validated targets for antimicrobial compounds. Recent approaches applying structure-based inhibitor design, high-throughput screening assays and whole cell assays have identified a large number of novel inhibitors of cytoskeletal proteins and enzymes of the peptidoglycan biosynthesis pathway.

2. The Outer Membrane of Gram-negative Bacteria: Lipopolysaccharide Biogenesis and Transport

Paola Sperandeo, Riccardo Villa, Gianni Dehò and Alessandra Polissi

The cell envelope of Gram-negative bacteria consists of two distinct membranes, the inner (IM) and the outer membrane (OM) separated by an aqueous compartment, the periplasm. The OM contains in the outer leaflet the lipopolysaccharide (LPS), a complex glycolipid with important biological functions. In the host, it elicits the innate immune response whereas in the bacterium it is responsible for the peculiar permeability barrier properties exhibited by the OM. LPS is synthesized in the cytoplasm and at the inner leaflet of the IM. It needs to cross two different compartments, the IM and the periplasm, to reach its final destination at the cell surface. In this chapter we will first summarize LPS structure, functions and biosynthetic pathway and then review in more details the studies that have led in the last decade to elucidate the protein machinery that ferries LPS from the IM to its final destination in the OM.

3. Outer Membrane Protein Biosynthesis: Transport and Incorporation of OM Proteins (in)to the OM bilayer

Kelly H. Kim, Suraaj Aulakh and Mark Paetzel

The outer membrane is a unique structural feature of Gram-negative bacteria. Within the outer membrane reside β-barrel outer membrane proteins that serve many important functions, such as nutrient uptake, virulence and cell signaling. Proper folding and assembly of these proteins are therefore essential for cell viability. Gram-negative bacteria possess a specialized proteinaceous machine known as the BAM (β-barrel assembly machinery) complex that is responsible for the proper assembly of β-barrel proteins into the outer membrane. This chapter summarizes the current status of knowledge about outer membrane protein biosynthesis, and the significant progress that has been made towards understanding the structure and function of the bacterial BAM complex.

4. Bacterial Lipoproteins; Biogenesis, Virulence/Pathogenicity and Trafficking

Hajime Tokuda, Peter Sander, Bok Luel Lee, Suguru Okuda, Thomas Grau, Andreas Tschumi, Juliane K. Brülle, Kenji Kurokawa and Hiroshi Nakayama

The mechanisms underlying the biogenesis and outer membrane sorting of lipoproteins have been mostly clarified in Escherichia coli. Three enzymes catalyze the post-translational modification of lipoproteins with a membrane anchor comprising a thioether-linked diacylglycerol and an amide linked fatty acid. The Lol system comprising five Lol proteins mediates the sorting of lipoproteins to the outer membrane. The three enzymes and five proteins are essential for E. coli and are widely conserved in Gram-negative diderms having cytoplasmic and outer membranes. High G+C content Gram-positive bacteria such as those belonging to the genera Mycobacterium and Corynebacterium have an outer membrane-like structure. The structure and biogenesis of the cell envelope have been intensively studied in mycobacterial species including medically important Mycobacterium tuberculosis. Mycobacterial lipoproteins are triacylated, like those of Gram-negative diderms. The enzyme catalyzing N-acylation was recently identified. The lipoprotein biosynthesis pathway is important for virulence of M. tuberculosis. The functions of individual mycobacterial lipoproteins are discussed in relation to envelope biogenesis, virulence and influence on immune systems. The N-terminal structures of lipoproteins of low G+C content Gram-positive monoderms are surprisingly diverse. An enzyme catalyzing the N-acylation of the N-terminal Cys of lipoproteins has not been found in this class of bacteria. However, lipoproteins of Staphylococcus species are N-acylated. Moreover, three novel structures of lipidated N-terminal Cys were revealed on mass spectrometric analyses. A possible biosynthetic pathway generating these structures is discussed.

5. The Fascinating Coat Surrounding Mycobacteria

Mamadou Daffé and Benoît Zuber

The mycobacterial cell envelope is fascinating in several ways. First, its composition is unique by the exceptional lipid content, which consists of very long-chain (up to C90) fatty acids, the so-called mycolic acids, and a variety of exotic compounds. Second, these lipids are atypically organized into a Gram-negative-like outer membrane (mycomembrane) in these Gram-positive bacteria, as recently revealed by CEMOVIS, and this mycomembrane also contains pore-forming proteins. Third, the mycolic acids esterified a holistic heteropolysaccharide (arabinogalacan), which in turn is linked to the peptidoglycan to form the cell wall skeleton (CWS). In slow-growing pathogenic mycobacterial species, this giant structure is surrounded by a capsular layer composed mainly of polysaccharides, primarily a glycogen-like glucan. The CWS is separated from the plasma membrane by a periplasmic space. A challenging research avenue for the next decade comprises the identification of the components of the uptake and secretion machineries and the isolation and biochemical characterization of the mycomembrane.

6. The Role Of Lipid Composition on Bacterial Membrane Protein Conformation and Function

Vinciane Grimard, Marc Lensink, Fabien Debailleul, Jean-Marie Ruysschaert and Cedric Govaerts

Cellular, biochemical and biophysical studies have shown over the last years that membrane proteins interact intimately with the lipid bilayer and that the structure and the activity of these proteins can be modulated by the type of lipids that surround them. Studies have demonstrated that physico-chemical properties of the membrane affect protein function. In fact some proteins directly sense the membrane properties, such as fluidity, tension, curvature stress or hydrophobic mismatch, for example as a signal for temperature or osmotic stress. In other cases, specific interactions between defined protein motifs and given lipids have been evidenced. These interactions often fulfill structural and functional role and are intimately linked to the biological function of membrane proteins. This chapter aims at summarizing the elaborate interplay existing between proteins and lipids and the influence of both specific interactions and bulk properties of the membrane on the stability, the structure and the activity of bacterial membrane proteins.

7. Bacterial ABC Transporters: Structure and Function

Anthony M. George and Peter M. Jones

ATP-Binding-Cassette (ABC) membrane transporters belong to one of the largest and most ancient gene families, occurring in bacteria, archaea, and eukaryota. In addition to nutrient uptake, ABC transporters are involved in other diverse processes such as the export of toxins, peptides, proteins, antibiotics, polysaccharides and lipids, and in cell division, bacterial immunity and nodulation in plants. While prokaryotic ABC transporters encompass both importers and exporters, eukaryotes harbour only exporters. Bacterial ABC transporters are intricately involved either directly or indirectly in all aspects of cellular physiology, metabolism, homeostasis, drug resistance, secretion, and cellular division. Whilst several complete ABC transporter structures have been solved over the past decade, their functional mechanism of transport is still somewhat controversial and this aspect is discussed in detail.

8. Energy-coupled Transport Across the Outer-membrane of Gram-negative Bacteria

Volkmar Braun

Active, energy-coupled transport across the Outer-membrane (OM) of Gram-negative bacteria is intriguing. Because there is no energy source in the periplasm, the energy required for transport is instead provided by the cytoplasmic membrane proton-motive force, to which a particular class of transport proteins responds. Such an energy-coupled transporter in the OM forms a β-barrel that is tightly closed on the periplasmic side by the N-terminal domain plug. This plug domain distinguishes active transporters from porins-other β-barrel proteins that form permanently open pores, through which substrates passively diffuse. The energy from the cytoplasmic membrane is transferred to the OM transporters by the TonB-ExbB-ExbD protein complex (Ton system). Crystal structures of transporters and of transporters with bound TonB fragments provide clues as to how OM transport is energized, but experimental evidence of how the Ton system responds to the proton motive force, how it transfers energy from the cytoplasmic membrane to the OM, and how the transporters react to energy input is largely lacking. Substrates actively taken up across the OM include heme, Fe3+ incorporated into siderophores, transferrin, lactoferrin, haptoglobin, hemopexin, lipocalin, vitamin B12, Ni2+, Zn2+, various oligosaccharides, and aromatic compounds. OM transporters also regulate transcription of genes from the cell surface and serve as receptors for toxic peptides and protein toxins. The transporters are particularly abundant in environmental and gut bacteria (up to 140 per strain), which use a large variety of substrates, but are absent in intracellular pathogens and obligate parasites.

9. The Permeability Barrier: Passive and Active Drug Passage Across Membranes

Kozhinjampara R Mahendran, Robert Schulz, Helge Weingart, Ulrich Kleinekathöfer and Mathias Winterhalter

Under antibiotic stress a reduced permeability for drugs to enter and an enhanced active extrusion of undesired compounds was observed in Gram-negative bacteria. With respect to the influx of drugs the first line of defense is the outer membrane containing a number of channel forming proteins called porins allowing passive penetration of water-soluble compounds into the periplasmic space. Mutations in the constriction zone or a reduction of the number of porins reduce the flux. In addition porins coupled to efflux pumps allow the cell to eject antibiotics actively into the extracellular space. In order to understand the function of the involved proteins, a quantification of the individual transport elements is necessary. Here we describe experimental and computational biophysical methods to characterize molecular transport of antibiotics and small compounds across bacterial membranes.

10. Targeting and Integration of Bacterial Membrane Proteins

Patrick Kuhn, Renuka Kudva, Thomas Welte, Lukas Sturm, and Hans-Georg Koch

Membrane proteins execute a plethora of essential functions in bacterial cells and therefore bacteria utilize efficient strategies to ensure that these proteins are properly targeted and inserted into the membrane. Most bacterial inner membrane proteins are recognized early during their synthesis, i.e. co-translationally by the bacterial signal recognition particle (SRP), which delivers the ribosome-nascent chain (RNC) via its interaction with the membrane-bound SRP receptor to the SecYEG translocon, a highly dynamic and evolutionarily conserved protein conducting channel. Membrane protein insertion via SecYEG is coupled to ongoing polypeptide chain elongation at the ribosome and the emerging transmembrane helices exit the SecYEG channel laterally into the lipid phase. Lateral release and folding of transmembrane helices is most likely facilitated by YidC, which transiently associates with the SecYEG translocon. YidC has also been shown to facilitate insertion of inner membrane proteins independently of the SecYEG translocon. The targeting of outer membrane proteins to SecYEG occurs predominantly post-translationally by the SecA/SecB pathway and thus follows the same route as periplasmic proteins. In this chapter, we summarize the current knowledge on membrane protein targeting and transport/integration by either SecYEG or YidC.

11. Envelope Spanning Secretion Systems in Gram-negative Bacteria

Matthias J Brunner, Rémi Fronzes and Thomas C Marlovits

Gram-negative bacteria have a cell envelope made of two membranes separated by a thin layer of peptidoglycan. To transport macromolecules such as proteins and DNA through the entire cell envelope, several types of secretion systems are employed. This chapter focuses on the structure and function of type III and type IV secretion systems. Type III secretion (T3S) systems are used by pathogens such as Salmonella, Shigella and Yersinia. They are composed of more than 20 different proteins, some of them present in multiple copies. Their function is to inject proteinaceous toxins, referred to as "effectors", into the eukaryotic host cell upon intimate contact. One pathogen typically secrets many different effectors-most of them have in common that they hijack part of the host cell machinery. Type IV secretion (T4S) systems are versatile secretion systems found in many bacteria. Typical T4S systems are made of 12 different proteins. Within the T4S family, three groups can be defined: First, T4S systems that mediate conjugative transfer of mobile genetic elements into a wide range of bacterial species but also into eukaryotic cells. Second, type IV secretion systems mediating DNA uptake or release from or into the extracellular milieu. And third, type IV secretion systems directly involved in virulence, secreting virulence factors into mammalian host cells. These are used by pathogens such as Helicobacter pylori or Legionella pneumophila.

12. Signaling Mechanisms in Prokaryotes

Mariano Martinez, Pedro M. Alzari and Gwénaëlle André-Leroux

Adaptation to an environmental stress is essential for cell survival in all organisms. In regard to this, the plasmatic membrane plays a fundamental role as it is the first barrier that separates the hostile environment from the cytoplasmic content, where all living reactions occur. Most of the machinery dedicated to sense upstream the external changes and to transduce the signals through the cytoplasmic membrane - upon which signals trigger a committed response dowstream - is located at this special interface. This chapter is dedicated to describing the two major signalling systems found in prokaryotes: the so-called two-component systems and the eukaryotic-like serine/threonine/tyrosine protein kinases. We will focus on the basic aspects of these sensing machineries, their function, catalytic mechanism and regulation, and will extend, when possible, to more sophisticated features, such as the structural basis of the signal sensing and transduction per se, based on the most recent investigations.

13. Outer-membrane-embedded and -associated Proteins and their Role in Adhesion and Pathogenesis

Vincent van Dam, Virginie Roussel-Jazédé, Jesús Arenas, Martine P. Bos and Jan Tommassen

The cell envelope of Gram-negative bacteria is composed of two membranes, which are separated by the periplasm containing a layer of peptidoglycan. The outer membrane is in contact with the environment. It contains a myriad of integral and associated proteins that are involved in adhesion to biotic and abiotic surfaces and, in the case of pathogens, in virulence. To reach the cell surface, these proteins have to cross the entire cell envelope, which is accomplished via various secretion pathways. Neisseria meningitidis, a commensal that lives in the nasopharynx but occasionally causes sepsis and/or meningitis, expresses a plethora of these virulence factors including type IV pili, proteins secreted via the type V secretion pathway, and cell surface-exposed lipoproteins. Here, we discuss the biogenesis and the function of such virulence factors with emphasis on those produced by N. meningitidis.

14. Bacterial Membranes as Drug Targets

Alvin Lo, Gaetano Castaldo and Han Remaut

Bacterial membranes play a pivotal role in maintaining cell integrity, in chemical energy generation and in the interplay between bacteria and their environment. Given their indispensible functions in bacterial physiology and metabolism, it comes as no surprise that bacterial membranes and membrane-associated biosynthetic and metabolic pathways form important targets for antibacterial compounds. Bacterial membranes and membrane-associated proteins and processes have formed the targets of natural antibiotics as well as promising starting points for the search and design of new synthetic antibacterials. In this chapter, we provide a review of existing examples of membrane-targeting antibiotics as well as an overview of the pathways that are currently under investigation for chemical knockdown.