Phospholipid modifications in bacteria

Many bacteria alter the acyl chains of their membrane phospholipids in response to changing environmental conditions. Two of these modifications are reviewed: cis-->trans isomerization and cyclopropanation of double bonds. The isomerization reaction is now known to be catalyzed by a periplasmic protein that contains a covalently bound heme. Cyclopropanation has been shown to play a role in Mycobacterium tuberculosis pathogenesis and, in Escherichia coli, plays an important role in resistance to acidic conditions.     

Phospholipid composition of methane-utilizing bacteria.

The phospholipids of Methylococcus capsulatus, Methylosinus trichosporium, La Paz, and OBT were examined in relation to their qualitative and quantitative composition. M. capsulatus exhibited a phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and phosphatidyl-choline. The esterified fatty acids were predominantly C16:0 and C16:1. M. trichosporium, La Paz, and OBT exhibited an essentially identical phospholipid composition consisting of phosphatidylmonomethylethanolamine, phosphatidyl-dimethylethanolamine, phosphatidylcholine, and phosphatidylglycerol. Only trace amounts (less than 1%) of cardiolipin were found in these organisms. The major esterified fatty acid in these organisms was C18:1 (87 to 90%). The monounsaturated fatty acids from all four organisms consisted of both cis and trans isomers, each of which contained delta8, delta9, delta10, and delta11 double-bond positional isomers.     

Regulation of directed motility in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the 'frizzy' signal-transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY-like response-regulator domains: one is present on FrzE, which also contains a CheA-like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self-generated autoattractants. The Frz signal-transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting-body development and vegetative swarming.     

Phospholipid biosynthesis in some anaerobic bacteria.

We have identified and characterized enzymes of phospholipid synthesis in two plasmalogen-rich anaerobes. Megasphaera elsdenii and Veillonella parvula, and one anaerobe lacking plasmalogens. Desulfovibrio vulgaris. All three species contained phosphatidate cytidylyltransferase and phosphatidylserine synthase. Phosphatidylglycerophosphate synthesis was detected only D. vulgaris extracts. Phosphatidylserine (diacyl form) was the major product of the phosphatidylserine synthase assay with particles from M. elsdenii or V. parvula. The amounts of phosphatidylethanolamine formed were very low. Only D. vulgaris particles had an active phosphatidylserine decarboxylase.     

Pseudomonas aeruginosa exhibits directed twitching motility up phosphatidylethanolamine gradients

Pseudomonas aeruginosa translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type P. aeruginosa, but not a pilJ pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing P. aeruginosa motility while many other species were inactive. P. aeruginosa PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in biofilm formation and pathogenesis.     

Hysteresis-based mechanism for the directed motility of the Ncd motor

Ncd is a Kinesin-14 family protein that walks to the microtubule's minus end. Although available structures show its α-helical neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck sequentially makes intermediate contacts with the motor head along its mostly longitudinal path, and it develops a 24° twist in the post-stroke orientation. The forward (pre-stroke to post-stroke) motion has an ∼4.5 k_BT (where k_B is the Boltzmann constant, and T = 300 K) free-energy barrier and is a diffusion guided by the intermediate contacts. The post-stroke free-energy minimum is higher and is formed ∼10° before reaching the orientation in the post-stroke crystal structure, consistent with previous structural data. The importance of intermediate contacts correlates with the existing motility data, including those for mutant Ncds. Unlike the forward motion, the recovery stroke goes nearly downhill in free energy, powered in part by torsional relaxation of the neck. The hysteresis in the energetics of the neck motion arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key to the directed motility of Ncd.     

Inducible directed evolution of complex phenotypes in bacteria

Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5–10 kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli’s utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.     

Egg jelly proteins stimulate directed motility in Xenopus laevis sperm

Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose‐dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488‐conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0–1 µg/ml range and correlated well with previously published dose‐dependent sperm attraction data. Binding was rapid with a half‐time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm‐orienting behavior. Mol. Reprod. Dev. 78:450–462, 2011. © 2011 Wiley‐Liss, Inc.     

Flagella and motility behaviour of square bacteria.

Square bacteria are shown to have right-handed helical (RH) flagella. They swim forward by clockwise (CW), and backwards by counterclockwise (CCW) rotation of their flagella. They are propelled by several or single filaments arising at several or single points on the cell surface. When there are several filaments a stable bundle is formed that does not fly apart during the change from clockwise to counterclockwise rotation or vice versa. In addition to the flagella attached to the cells, large amounts of detached flagella aggregated into thick super-flagella, can be observed at all phases of growth.     

High motility reduces grazing mortality of planktonic bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 microm s(-1) as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 microm s(-1)) and a highly motile strain (>45 microm s(-1)) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 microm(3). Grazing mortality was lowest for cells of >0.5 microm(3) and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (< or =0.1 microm(3), >50 microm s(-1)) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

Use of a computer to assay motility in bacteria

An assay was developed to study the movement of free-swimming Escherichia coli. Cells were videotaped through a microscope, and the videotape images were then digitized and analyzed with a computer. Angular and linear speeds were measured for wild-type E. coli and for a smooth and a tumbly mutant. The average angular and linear speeds of a population were directly and inversely proportional, respectively, to the time spent tumbling. Changes in angular and linear speeds were followed during the response of wild-type E. coli to attractant or repellent.     

Phospholipid and acid composition of Nocardia and nocardoid bacteria as criteria of classification

Phospholipid and acid composition of 5 strains of ‘true’ Nocardia and 4 strains of nocardoid bacteria have been studied. A great homogeneity was found in all the Nocardia species: phospholipids consist of cardiolipin, phosphatidyl ethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Streptomyces (Nocardia) mediterranei did not contain phosphatidylinositol and Oerskovia (Nocardia) turbata had no phosphatidyl ethanolamine. The fatty acid composition of these phospholipids was determined and was found different in Nocardia and nocardoid species. Nocardia were rich in straight chain fatty acids and tuberculostearic acid while the phospholipids of nocardoid bacteria contained greater amounts of branched fatty acids. The fatty acids from acetone soluble lipids consisted of hydroxy and non-hydroxy compounds. Hydroxy acids were found in Nocardia which contained nocardic acids: high MW β-hydroxy α-branched acids and in S. mediterranei which contained β-hydroxy acids with 15–17 carbon atoms. Non-hydroxy acids were essentially palmitic and tuberculostearic acids in Nocardia species while S. mediterranei and O. turbata contained great amounts of iso acids from C₁₄ to C₁₇. Phospholipid and acid composition are discussed as criteria of taxonomic classification of Nocardia and related Actinomycetes.     

Phospholipid fatty acid and lipopolysaccharide fatty acid signature lipids in methane-utilizing bacteria

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.     

Phospholipid composition and cardiolipin synthesis in fermentative and nonfermentative marine bacteria.

Twenty biochemically distinct isolates of marine bacteria, comprising a collection of gram-negative, motile, straight and curved rod-shaped organisms, were separated into fermentative and nonfermentative groups. The isolates were analyzed fro phospholipid composition and the activities of the enzymes, cardiolipin synthetase, and a phosphilipase were determined. The phospholipid compositions of all isolates were generally similar. Phosphatidylethanolamine and phosphatidylglycerol were the major phospholipid classes detected. The absence of cardiolipin in most of the nonfermentative isolates was the most striking observation noted. This was verified chromatographically and by the absence of cardiolipin synthetase activity. In isolates which had cardiolipin, it apparently was synthesized by the condensation of two molecules of phosphatidylglycerol, a mechanism similar to that observed in terrestrial bacteria. Possible correlations between the presence of cardiolipin and Mg-2+ requirements for growth are discussed.     

A laser-diffraction capillary assay to measure random motility in bacteria

A laser-diffraction capillary assay was developed to measure bacterial random motility coefficients in semi-solid media. A capillary was filled successively with two agar suspensions, one of which contained bacteria. A laser was directed axially through the capillary. Random motility coefficients were calculated for Pseudomonas stutzeri KC and Escherichia coli at different agar concentrations.     

Emergence and modular evolution of a novel motility machinery in bacteria

Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena.     

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus‐ends in an EB‐dependent manner or moving processively towards minus ends in an adaptor protein‐dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal‐D2 (BicD2) or the multifunctional regulator Lissencephaly‐1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus‐end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.