Genetic and mass spectrometry analyses of the unusual type IV-like pili of the archaeon Methanococcus maripaludis

The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.     

Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus , T4P are unipolarly localized and undergo pole-to-pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non-piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole-to-pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP–PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

New type of organic matrix in cirals formed at the decalcified site: Structure and composition

This is the first demonstration that the organic matrix appears at the decalcified site of the skeleton in a juvenile coral of Fungia fungites (Linnaeus). This matrix was secreted by the calicoblastic layer and is composed of fibrous matters as seen in mesoglea. If formed a thick sheet at the advanced stage. The amino acid composition of this matrix was similar to that in the skeleton rather than mesogleal protein. Morphologic and functional features suggest that the organic sheet must be similar to the sheet which is secreted extracellularly by planula larva at the time of settlement. The sheet in F. fungites may have a role of protecting calicoblastic epithelium from exposure as a result of skeletal dissolution.     

Molecular cloning, characterization and localization of chicken type II procollagen gene

Chicken type II procollagen (ccol2a1) has become as an important oral tolerance protein for effective treatment of rheumatoid arthritis. However, its molecular identity remains unclear. Here, we reported the full-length cDNA and nearly complete genomic DNA encoding ccol2a1. We have determined the structural organization, evolutional characters, developmental expression and chromosomal mapping of the gene. The full-length cDNA sequence spans 4837 bp containing all the coding region of the ccol2a1 including 3' and 5' untranslation region. The deduced peptide of ccol2a1, composed of 1420 amino acids, can be divided into signal peptide, N-propeptide, N-telopeptide, triple helix, C-telopeptide and C-propeptide. The ccol2a1 genomic DNA sequence was determined to be 12,523 bp long containing 54 exons interrupted by 53 introns. Comparison of the ccol2a1 with its counterparts in human, mouse, canine, horse, rat, frog and newt revealed highly conserved sequence in the triple helix domain. Chromosomal mapping of ccol2a1 locates it on 4P2. While the ccol2a1 mRNA was expressed in multiple tissues, the protein was only detected in chondrogenic cartilage, vitreous body and cornea. The ccol2a1 was found to contain two isoforms detected by RT-PCR. The distribution of the ccol2a1 lacking exon 2wasfrequently detected in chondrogenic tissues, whereas the exon 2-containing isoform was more abundant in non-chondrogenic tissues. These results provide useful information for preparing recombinant chicken type II collagen and for a better understanding of normal cartilage development.     

Nucleotide sequence, regulation and functional analysis of the papC gene required for cell surface localization of Pap pili of uropathogenic Escherichia coli

The papC gene of uropathogenic Escherichia coli is required for the formation of digalactoside-binding Pap pili. papC forms part of an operon wherein the regulatory gene papB, the major pilin gene papA, a minor pilin-like gene papH, and papC are co-transcribed. Furthermore, the extent of PapC synthesis was found to affect the number of pili expressed on the cell surface. The DNA sequence of the papC gene is presented and its deduced amino acid sequence is compared to that of the FaeD protein encoded by the K88 pili gene cluster. The PapC protein was localized to the E. coli outer membrane where it may form a trans-membrane channel through which pilin subunits are surface localized.     

Mechanical removal of F pili, type I pili, and flagella from Hfr and RTF donor cells and the kinetics of their reappearance

The effect of mechanical agitation (blending) on the removal of F pili, type I pili, and flagella from Hfr (high-frequency recombinant) and resistance transfer factor (RTF) fi(+)Escherichia coli cells was studied by electron microscopy. The reduction in number and length of appendages was measured as a function of blendor speed under standard conditions of temperature, medium, cell density, and blendor configuration. F pili and flagella were removed within the same narrow range of blendor speeds. Type I pili were removed within a higher and broader range of speeds. The speed which reduced the average length of type I pili to 50% was 3.5 times the speed which reduced the average length of F pili to 50%. None of the speeds employed inhibited cell growth, viability, or the ability to produce cell appendages. The kinetics of reappearance of F pili and type I pili after removal by blending were also different. F pili grew out very rapidly, reaching 50% of their full length in 30 sec and their full length in 4 to 5 min. The number of attached F pili per cell also increased rapidly, reaching a constant value in 4 to 5 min. After 5 min, F pilus lengths were distributed around a modal value of about 1.2 mum, and the numbers of F pili per cell were distributed according to a Poisson distribution, with an average of 1.0 per cell. These reappearance kinetics, length distributions, and number distributions are consistent with a model of F-pilus outgrowth in which new F pili appear at random locations on the cell surface at an average rate of about once every 4 min, grow to their characteristic length in about 4 min, and then separate from the cell. F pili which had separated could absorb to the cells, leading to the presence of two classes of F pili on cells: those in the process of natural out-growth and those attached by absorption. Type I pili increased in length much more slowly than did F pili, although the fraction of cells having visible type I pili increased very rapidly after blending because of the large number of type I pili per cell. The fraction of flagellated cells increased even more slowly, reaching only 30% of the unblended fraction in 30 min. The application of blending spectra and reappearance kinetics to the identification of cell functions with surface structures is discussed.     

A new way out: protein localization on the bacterial cell surface via Tat and a novel Type II secretion system

The ability to move proteins out of the cytoplasm and across membranes is a key aspect of the physiology and pathogenicity of Gram-negative bacteria. In this issue of Molecular Microbiology , Ferrandez and Condemine describe a novel protein targeting system in the enteric phytopathogen, Dickeya dadantii . The pectin lyase, PnlH, is exported by the Tat system and is somehow targeted to the outer membrane by its uncleaved N-terminal Tat signal anchor. A novel Type II secretion system, Stt, is then responsible for moving it across the outer membrane, where it remains localized on the surface of the cell. We discuss the implications of these findings for our understanding of both the mechanisms and physiological importance of bacterial protein targeting.     

Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.     

Influence of pili, fibrils, and capsule on in vitro adherence by Haemophilus influenzae type b

Haemophilus influenzae type b is an encapsulated bacterium that initiates infection by colonizing the upper respiratory epithelium. In vitro studies indicate that H. influenzae type b is capable of expressing two morphologically distinct filamentous adhesive structures, referred to as pili and fibrils, respectively. In this study, we examined adherence to a variety of human epithelial-cell types and demonstrated that pili and fibrils have separate cellular binding specificities. In addition, we found that capsular material inhibits fibril recognition of the host-cell surface. This inhibitory effect was reduced when bacteria were grown to stationary phase, reflecting diminished encapsulation. However, when growth medium was supplemented with Mg²⁺, stationary-phase organisms were relatively heavily encapsulated and non-adherent. These observations suggest that encapsulation can be modulated in response to growth phase or environmental signals. It is possible that encapsulation is down-modulated early in the infectious process in order to avoid interfering with colonization. In contrast, encapsulation may be up-modulated between hosts and during bacteremia, where it appears to confer a selective advantage. We speculate that this model may also apply to other encapsulated pathogens.     

The Cys-67 residue of HLA-B27 influences cell surface stability, peptide specificity, and T-cell antigen presentation.

Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 --> Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705- and Cys-67 --> Ser-bound peptides revealed that the mutant failed to bind approximately 15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 --> Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 --> Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy.     

FTIR spectroscopy of complexes formed between metarhodopsin II and C-terminal peptides from the G-protein alpha- and gamma-subunits

Metarhodopsin II (MII) provides the active conformation of rhodopsin for interaction with the G-protein, Gt. Fourier transform infrared spectra from samples prepared by centrifugation reflect the pH dependent equilibrium between MII and inactive metarhodopsin I. C-terminal synthetic peptides (Gtalpha(340-350) and Gtgamma(60-71)farnesyl) stabilize MII. We find that both peptides cause similar spectral changes not seen with control peptides (Gtalpha (K341R, L349A) and non-farnesylated Gtgamma). The spectra reflect all the protonation dependent bands normally observed when MII is formed at acidic pH. Beside the protonation dependent bands, additional features, similar with both peptides, appear in the amide I and II regions.     

Electron-microscopic localization of type II,IX, and V collagen in the organ of Corti of the gerbil

Summary The presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.     

Purification and polar localization of pneumococcal LytB,a putative endo-beta-N-acetylglucosaminidase: the chain-dispersing murein hydrolase

The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in Escherichia coli. LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-beta-N-acetylglucosaminidase of Streptococcus pneumoniae. LytB can remove a maximum of only 25% of the radioactivity from [(3)H]choline-labeled pneumococcal cell walls in in vitro assays. Inactivation of the lytB gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence. Longer chains were formed when the lytB mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of lytA, which codes for the major autolysin. Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus. Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively. In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced. On the other hand, the use of a translational fusion between the gene (gfp) coding for the green fluorescent protein (GFP) and lytB supports the notion that LytB accumulates in the cell poles of either the wild-type R6, lytB mutants, or ethanolamine-containing cells (EA cells). The GFP-LytB fusion protein was also able to unchain the lytB mutants but not the EA cells. In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length. These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells.     

Interaction of tyrosyl, histidyl, and tryptophanyl peptides with DNA: specificity and mechanism of the interaction

In the Tyr-(Gly)_1-4-Tyr series maximal thermal stabilization of calf thymus DNA (δT_m=10°) occurred with the Tyr-(Gly)₂-Tyr peptide, where three base pairs could separate the two tyrosyl residues. Tyr-Gly-Tyr-Gly-Tyr stabilized the DNA by 6°. The alternating Trp-Gly-Trp-Gly-Trp and His-Gly-His-Gly-His peptides were equally as effective as the Tyr-Gly-Tyr-Gly-Tyr peptide in stabilizing calf thymus DNA against thermal denaturation. But the alternating Phe-Gly-Phe-Gly-Phe peptide afforded little stabilization, suggesting that a sidechain possessing both a conjugated π-electron system and an electron donor atom is necessary for DNA stabilization. Introduction of electron withdrawing iodo or nitro group into the tyrosyl sidechains almost completely abolished the stabilizing effect. Although the tyrosyl peptides seem to be specific for GC-base pairs, no correlation was found in natural DNA between% GC and% thermal stabilization. Eukaryotic DNAs showed twice the stabilization of prokaryotic DNAs with the same GC content.