Towards a structural biology of bacterial conjugation

Horizontal DNA transfer among bacteria (conjugation) requires the formation of secure intercellular contacts before DNA transfer can occur. The formation of such contacts among Gram-negative bacteria is mediated by conjugative pili. Like other pili, conjugative pili are filaments extending from the surface of donor cells. They are composed, in so far as is known, entirely of conjugative pilin subunits. Here, we review the structure of F-pilin, the subunit of which F-pili are composed. We emphasize recent studies suggesting a specific domain organization for F-pilin and present a model of how these domains might be arranged in filament subunits.     

Contacts between the cells in bacterial colonies

The interaction of cells in microbial colonies has been studied by electron-microscopic techniques. Two types of contacts between cells have been found to exist in the colonies of Gram-negative bacteria of the genera Escherichia, Shigella and Salmonella: close cell adhesion due to the fusion of cell-wall outer membranes and the formation of intersections consisting of membranous tubules. At the sites of close adhesion the fusion of cytoplasmic and outer membranes have been found to occur in Bayer's zones. In the colonies of Gram-positive bacteria of the genera Staphylococcus and Brevibacterium only one type of contacts has been revealed: the fusion of the peptidoglycan layers of the cell walls. The results of this study indicate that in colonies bacteria are not completely isolated; their interaction leads to the formation of a three-dimensional structure denoted as a cooperative cell system.     

Enterococcus adhesin PrgB facilitates type IV secretion by condensation of extracellular DNA

Conjugative type IV secretion systems (T4SSs) are multi‐protein complexes in Gram‐negative and Gram‐positive (G+) bacteria, responsible for spreading antibiotic resistances and virulence factors among different species. Compared to Gram‐negative bacteria, which establish close contacts for conjugative transfer via sex pili, G+ T4SSs are suggested to employ surface adhesins instead. One example is pCF10, an enterococcal conjugative sex‐pheromone responsive plasmid with a narrow host range, thus disseminating genetic information only among closely related species. This MicroCommentary is dedicated to the crystal structure of the pCF10‐encoded adhesion domain of PrgB presented by Schmitt et al. The authors show in their work that this adhesion domain is responsible for biofilm formation, tight binding and condensation of extracellular DNA (eDNA) and conjugative transfer of pCF10. A sophisticated two‐step mechanism for highly efficient conjugative transfer is postulated, including the formation of PrgB‐mediated long‐range intercellular contacts by binding and establishment of shorter‐range contacts via condensation of eDNA. PrgB binding to lipoteichoic acid on the recipient cell surface stabilizes junctions between the mating partners. The major findings by Schmitt et al. will be brought into a broader context and potential medical applications targeting eDNA as essential component in biofilm formation and conjugation will be discussed.     

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome

In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation.     

Structural basis for the antibiotic activity of ketolides and azalides

The azalide azithromycin and the ketolide ABT-773, which were derived by chemical modifications of erythromycin, exhibit elevated activity against a number of penicillin- and macrolide-resistant pathogenic bacteria. Analysis of the crystal structures of the large ribosomal subunit from Deinococcus radiodurans complexed with azithromycin or ABT-773 indicates that, despite differences in the number and nature of their contacts with the ribosome, both compounds exert their antimicrobial activity by blocking the protein exit tunnel. In contrast to all macrolides studied so far, two molecules of azithromycin bind simultaneously to the tunnel. The additional molecule also interacts with two proteins, L4 and L22, implicated in macrolide resistance. These studies illuminated and rationalized the enhanced activity of the drugs against specific macrolide-resistant bacteria.     

An automatic method for the measurement of the effect of short stresses on the microbial ATP pool

An automatic method for measuring the effects of brief contacts between microorganisms and toxic agents is described.In this method, both a microbial culture and a toxic solution are injected into a mixing tube, until inhibition of bacteria and ATP extraction take place.The range of the contact times, defined as the residence periods of the cultures in the mixing tube, is quite wide (from 0.3 to 16 s), with increased coefficient of variation (from 5 to 14%) for the lower times.     

Listeria monocytogenes Exploits Normal Host Cell Processes to Spread from Cell to Cell✪

The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1-2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.     

Mechanisms of polar arrest of a replication fork

A DNA replication terminator sequence blocks an approaching replication fork when the moving replisome approaches from just one direction. The mechanism underlying polar arrest has been debated for years, but recent work has helped to reveal how a replication fork is blocked in Escherichia coli . Early work suggested that asymmetric interaction between terminator protein and terminator DNA contributes to polar fork arrest. A later study demonstrated that if the terminator DNA is partially unwound, the resulting melted DNA could bind tightly to the terminator protein, suggesting a mechanism for polar arrest that involves a locked complex. However, recent evidence suggests that the terminator protein–DNA contacts are not sufficient for polar arrest in vivo . Furthermore, polar arrest of a replication fork still occurs in the absence of a locked complex between the terminator protein and DNA. In E. coli and Bacillus subtilis , the bound terminator protein makes protein–protein contacts with the replication fork helicase, and these contacts are critical in blocking progression of the advancing fork. Thus, we propose that interactions between the replication fork helicase and terminator protein are the primary mechanism for polar fork arrest in bacteria, and that this primary mechanism is modulated by asymmetric contacts between the terminator protein and its cognate DNA sequence. In yeast, terminator sequences are present in rDNA non-transcribed spacers and a region immediately preceding the mating type switch locus Mat1, and the mechanism of polar arrest at these regions is beginning to be elucidated.     

pH-induced structural changes of apo-lactoferrin and implications for its function: a molecular dynamics simulation study

The minor protein in milk, lactoferrin (Lf), is known for a variety of biological functions, and has been investigated as a protective encapsulant for probiotic bacteria in health-promoting food products. Lf is likely to be exposed to extreme pH conditions which are known to have disruptive influences on its functionality. The molecular mechanisms underlying these pH-dependent changes are not well-understood. To explore the potential of Lf as an encapsulant, molecular dynamics (MD) simulations were applied to study its conformational changes under extreme acidic (pH 1.0) or basic (pH 14.0) conditions, relative to neutral pH. Simulations indicate that the structure of apo-Lf is relatively stable at neutral pH, while acidic and basic pH result in substantially greater flexibility, partly induced by the loss of contacts between the N- and C-terminal lobes, causing them to undergo extensive relative bending and twisting motions. Basic pH causes greater structural disruption compared to acidic exposure. The latter has greater influence on the N-terminus, with increased fluctuations and disruptions of inter-residue contacts compared to those at neutral pH; while basic pH was found to more prominently disrupt contacts at the C-terminus. These results help elucidate possible functional consequences on Lf of exposure to extreme pH conditions.     

Circulation flow of enteropathogenic bacteria in institutionalized children. I. An epidemiologic study

The circulation flow and maintenance of enteropathogenic bacteria were studied from May 1982 to April 1983 in a population of institutionalized children and adult staff contacts in the city of Rio de Janeiro, Brazil. Subjects were assigned to three groups: A and B, included, respectively, 105 and 46 children with diarrhea who were admitted in the institution in different periods, and group C with 82 adult contacts. Faecal cultures were positive in 35.2%, 39.1% and 19.7% of subjects of groups A, B, and C, respectively. It suggests that the transmission was probably fostered by the environment because of as high as 30% of faecal contamination was found in environmental samples. Higher rate of isolation and elevated antibodies levels pointed out that Escherichia coli (EPEC) was the prevalent agent. Shigella predominated in the serological tests. These findings suggest that the institution itself may play an important role in the epidemiology and transmission of enteric infections in the community.     

YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.     

Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

The homologous hexameric AAA⁺ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.     

Impact of the microscale distribution of a Pseudomonas strain introduced into soil on potential contacts with indigenous bacteria

Soil bioaugmentation is a promising approach in soil bioremediation and agriculture. Nevertheless, our knowledge of the fate and activity of introduced bacteria in soil and thus of their impact on the soil environment is still limited. The microscale spatial distribution of introduced bacteria has rarely been studied, although it determines the encounter probability between introduced cells and any components of the soil ecosystem and thus plays a role in the ecology of introduced bacteria. For example, conjugal gene transfer from introduced bacteria to indigenous bacteria requires cell-to-cell contact, the probability of which depends on their spatial distribution. To quantitatively characterize the microscale distribution of an introduced bacterial population and its dynamics, a gfp-tagged derivative of Pseudomonas putida KT2440 was introduced by percolation in repacked soil columns. Initially, the introduced population was less widely spread at the microscale level than two model indigenous functional communities: the 2,4-dichlorophenoxyacetic acid degraders and the nitrifiers (each at 10(6) CFU g(-1) soil). When the soil was percolated with a substrate metabolizable by P. putida or incubated for 1 month, the microscale distribution of introduced bacteria was modified towards a more widely dispersed distribution. The quantitative data indicate that the microscale spatial distribution of an introduced strain may strongly limit its contacts with the members of an indigenous bacterial community. This could constitute an explanation to the low number of indigenous transconjugants found most of time when a plasmid-donor strain is introduced into soil.     

Biological role of bacterial inclusion bodies: a model for amyloid aggregation

Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.     

The epidemiology of neonatal meningitis

The epidemiological features of meningitides in newborns have been studied and criteria making it possible to distinguish the cases of intrauterine and postnatal infection have been proposed. The overwhelming majority of these infections has been found to occur due to hospital infections caused by enterobacteria and nonfermenting bacteria. In meningitides of this etiology the sources of infection are newborns colonized by these microorganisms. Infection is transmitted through everyday contacts through the hands of the medical staff. Besides, moist environmental objects serve as the additional reservoir of infection.     

Polymyxin B-lipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study

The dramatically increased frequency of antibiotic resistance has led to intensive efforts towards developing new families of antibiotics. Membrane-active antibiotic peptides such as polymyxin B (PxB) hold promise as the next generation of antibiotics, since they rarely spur the evolution of resistance. At low concentrations in the membrane, PxB forms vesicle-vesicle contacts and induces lipid exchange without leakage or fusion, a phenomenon that can explain its specificity towards gram-negative bacteria by contact formation between the two phospholipids interfaces in the periplasmatic space. In this work, the interaction of PxB and the nonantibiotic derivative polymyxin B nonapeptide (PxB-NP) with monolayers of Escherichia coli membrane lipids (ECL) has been studied by thermodynamic and structural methods. PxB inserts itself into ECL monolayers as a conformation that forms intermembrane contacts with vesicles injected underneath, and induces lipid exchange when the monolayer surface pressure is set at 32 mN/m (membrane equivalence pressure) or net transfer vesicle-to-monolayer at lower surface pressures. Thermodynamic analysis of the compression isotherms of mixed monolayers indicates that PxB inserts into the monolayer with an expansion of the mean molecular area, implying that peptide and lipids form nonideal mixtures. At low concentrations, corresponding to the membrane-membrane contact form of PxB, the mixed monolayers present positive excess energy values (deltaGm(Ex)), and atomic force microscopy (AFM) imaging reveals structures of approximately 120-nm diameter that protrude from the lipid surface approximately 0.7 nm. At concentrations of PxB above 4 mol %, thermodynamic analysis gives a very high deltaGm(Ex), corresponding to nonfavorable interactions, and AFM images show round structures of 20-30 nm diameter. PxB-NP behaves in a totally different way, in agreement with its inability to form vesicle-vesicle contacts and its lack of antibiotic effect. These results are discussed in the light of the mechanism of action of PxB on the membrane of gram-negative bacteria.     

The leading routes and factors in the transmission of the causative agent of campylobacteriosis under current conditions]

Epidemiological investigations carried out at the foci of Campylobacter infection in Moscow and the Moscow region in 1987-1990 demonstrated that Campylobacter infection was recently registered as sporadic cases in a few foci. The alimentary route of the transmission of this infection was the main factor of its spread. A high role of everyday contacts in the spread of this infection was noted. The possibility for outbreaks and sporadic cases of Campylobacter infection to be masked by very frequently occurring associations of these bacteria is discussed.     

Differences in Contacts of RNA Polymerases from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus aquaticus</Emphasis> with <Emphasis Type="Italic">lac</Emphasis>UV5 Promoter Are Determined by Core-Enzyme of RNA Polymerase

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.     

Close encounters: contact-dependent interactions in bacteria

Bacterial cells interact extensively within and between species. These interactions can be divided into those that rely on diffusible factors and those that depend on direct cell-to-cell contacts. An example of a contact-dependent interaction is the transfer of lipoproteins between Myxococcus xanthus cells that leads to transient stimulation of motility in certain motility mutants. In this issue of Molecular Microbiology, Wei et al. (2011) provide mechanistic insights into this contact-dependent transfer of lipoproteins. Briefly, a heterologous protein fused to a type II (lipoprotein) signal sequence that targets the protein to the outer membrane is required and sufficient for transfer. Moreover, evidence is provided that transfer may depend on specific contacts between donor and recipient cells. The data demonstrate that lipoprotein transfer in M. xanthus is not restricted to a few odd motility proteins but could be a wide-spread phenomenon in M. xanthus and possibly other bacteria. Recent years have been fruitful in identifying contact-dependent interactions between bacterial cells. These interactions can be grouped into those that involve delivery of cargo to a recipient and those that seem to be involved in cell-to-cell signalling. Several contact-dependent interactions involve widely conserved proteins, suggesting that cell contact-dependent processes may be widespread among bacteria.     

Impact of the Microscale Distribution of a Pseudomonas Strain Introduced into Soil on Potential Contacts with Indigenous Bacteria

Soil bioaugmentation is a promising approach in soil bioremediation and agriculture. Nevertheless, our knowledge of the fate and activity of introduced bacteria in soil and thus of their impact on the soil environment is still limited. The microscale spatial distribution of introduced bacteria has rarely been studied, although it determines the encounter probability between introduced cells and any components of the soil ecosystem and thus plays a role in the ecology of introduced bacteria. For example, conjugal gene transfer from introduced bacteria to indigenous bacteria requires cell-to-cell contact, the probability of which depends on their spatial distribution. To quantitatively characterize the microscale distribution of an introduced bacterial population and its dynamics, a gfp-tagged derivative of Pseudomonas putida KT2440 was introduced by percolation in repacked soil columns. Initially, the introduced population was less widely spread at the microscale level than two model indigenous functional communities: the 2,4-dichlorophenoxyacetic acid degraders and the nitrifiers (each at 10⁶ CFU g⁻¹ soil). When the soil was percolated with a substrate metabolizable by P. putida or incubated for 1 month, the microscale distribution of introduced bacteria was modified towards a more widely dispersed distribution. The quantitative data indicate that the microscale spatial distribution of an introduced strain may strongly limit its contacts with the members of an indigenous bacterial community. This could constitute an explanation to the low number of indigenous transconjugants found most of time when a plasmid-donor strain is introduced into soil.