The β-barrel assembly machinery (BAM) is required for the assembly of a primitive S-layer protein in the ancient outer membrane of Thermus thermophilus

The ancient bacterial lineage Thermus spp has a primitive form of outer membrane attached to the cell wall through SlpA, a protein that shows intermediate properties between S-layer proteins and outer membrane (OM) porins. In E. coli and related Proteobacteria, porins are secreted through the BAM (β-barrel assembly machinery) pathway, whose main component is BamA. A homologue to this protein is encoded in all the Thermus spp so far sequenced, so we wondered if this pathway could be responsible for SlpA secretion in this ancient bacterial model. To analyse this hypothesis, we attempted to get mutants on this BamA^th of T. thermophilus HB27. Knockout and deletion mutants lacking the last 10 amino acids were not viable, whereas its depletion by means of a BamA antisense RNA lead defective attachment to the cell wall of its OM-like envelope. Such defects were related to defective folding of the SlpA protein that was more sensitive to proteases than in a wild-type strain. A similar phenotype was found in mutants lacking the terminal Phe of SlpA. Further protein–protein interaction assays confirmed the existence of specific binding between SlpA and BamA^th. Taking together, these data suggest that SlpA is secreted through a BAM-like pathway in this ancestral bacterial lineage, supporting an ancient origin of this pathway before the evolution of the Proteobacteria.     

Homogeneous incorporation of secondary cell wall polysaccharides to the cell wall of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27

Regular surface protein layers (S-layers) from most Gram-positive bacteria and from the ancestral bacterium Thermus thermophilus attach to pyruvylated polysaccharides (SCWP) covalently bound to the peptidoglycan through their SLH domain. However, it is not known whether the synthesis of SCWP and S-layer is coordinated enough as to follow a similar pattern of incorporation to the cell wall during growth. In this work we analyse the localization of newly synthesized SCWP on the cell wall of T. thermophilus by immunoelectron microscopy. For this, we obtained mutants with a reduced amount of pyruvylated SCWP through mutation of the csaB gene encoding the SCWP-pyruvylating activity, and its upstream gene csaA, a putative sugar transporter. We hypothesized that CsaA would be required for the synthesis of the SCWP. However, we found that csaA mutants showed only a minor decrease in the amount of SCWP immunodetected on the cell walls in comparison with csaB mutants, revealing its irrelevance in the process. Complementation experiments of csaB mutants with CsaB expressed from inducible promoters revealed that newly synthesized SCWP was homogeneously distributed along the cell wall. Fusions with thermostable fluorescent protein revealed that CsaB was distributed also in homogeneous pattern associated with the membrane. These data support that synthesis of SCWP takes place in disperse and homogeneous form all over the cell surface, in contrast to the zonal incorporation at the cell centre recently demonstrated for SlpA.     

On the S-layer of Thermus thermophilus and the assembling of its main protein SlpA

We have isolated and analysed the cell envelope of the thermophilic bacterium Thermus thermophilus HB8. Isolated cell walls, characterized by the dominance of the S-layer protein SlpA, are found to be constituted by several protein complexes of high molecular weights. Further isolation steps, starting from the cell wall samples, led to the selective release of the S-layer protein SlpA in solution as confirmed by mass spectrometry. Blue Native gel electrophoresis on these samples showed that SlpA is organized into a specific hierarchical order of oligomeric states that are consistent with the complexes at high molecular weight identified on the total cell wall fraction. The analysis showed that SlpA bases this peculiar organization on monomers and exceptionally stable dimers, leading to the formation of tetramers, heptamers, and decamers. Furthermore, the two elementary units of SlpA, monomers and dimers, are regulated by the presence of calcium not only for the assembling of monomers into dimers, but also for the splitting of dimers into monomers. Finally, the SlpA protein was found to be subjected to specific proteolysis leading to characteristic degradation products. Findings are discussed in terms of S-layer assembling properties as bases for understanding its structure, turn-over and organization.     

Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII

Gram‐positive surface proteins can be covalently or non‐covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non‐identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock‐down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram‐positive bacteria.     

Functionality of the S-layer protein from the probiotic strain <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> M92

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

Bacterial outer membrane constriction

The outer membrane of Gram‐negative bacteria is a crucial permeability barrier allowing the cells to survive a myriad of toxic compounds, including many antibiotics. This innate form of antibiotic resistance is compounded by the evolution of more active mechanisms of resistance such as efflux pumps, reducing the already limited number of clinically relevant treatments for Gram‐negative pathogens. During cell division Gram‐negative bacteria must coordinate constriction of the outer membrane in conjunction with other crucial layers of the cell envelope, the peptidoglycan cell wall and the inner membrane. Coordination is crucial in maintaining structural integrity of the envelope, and represents a highly vulnerable time for the cell as any failure can be fatal, if not least disadvantageous. However, the molecular mechanisms of cell division and how the biogenesis of the three layers is synchronised during constriction remain largely unknown. Perturbations of the outer membrane have been shown to increase the effectiveness of antibiotics in vitro, and so with improved understanding of this process we may be able to exploit this vulnerability and improve the effectiveness of antibiotic treatments. In this review the current knowledge of how Gram‐negative bacteria facilitate constriction of their outer membranes during cell division will be discussed.     

A PopZ‐linked apical recruitment assay for studying protein–protein interactions in the bacterial cell envelope

Most bacteria are surrounded by a complex cell envelope. As with many biological processes, studies of envelope assembly have benefited from cell‐based assays for detecting protein–protein interactions. These assays use simple readouts and lack a protein purification requirement, making them ideal for early stage investigations. The most widely used two‐hybrid interaction assay for proteins involved in envelope biogenesis is based on the reconstitution of adenylate cyclase activity from a split enzyme. Because adenylate cyclase is only functional in the cytoplasm, both protein fusions used in the assay must have a terminus located in this compartment. However, many envelope assembly factors are wholly extracytoplasmic. Detecting interactions involving such proteins using two‐hybrid systems has therefore been problematic. To address this issue, we developed a cytological assay in Escherichia coli based on PopZ from Caulobacter crescentus. Here, we demonstrate the utility of this PopZ‐Linked Apical Recruitment (POLAR) method for detecting interactions between proteins located in different cellular compartments. Additionally, we report that recruitment of an active peptidoglycan synthase to the cell pole is detrimental for E. coli and that interactions between proteins in the inner and outer membranes of the Gram‐negative envelope may provide a mechanism for recruiting protein complexes to subpolar sites.     

A new factor stimulating peptidoglycan hydrolysis to separate daughter cells in Caulobacter crescentus

Cell division in Gram‐negative bacteria involves the co‐ordinated invagination of the three cell envelope layers to form two new daughter cell poles. This complex process starts with the polymerization of the tubulin‐like protein FtsZ into a Z‐ring at mid‐cell, which drives cytokinesis and recruits numerous other proteins to the division site. These proteins are involved in Z‐ring constriction, inner‐ and outer‐membrane invagination, peptidoglycan remodelling and daughter cell separation. Three papers in this issue of Molecular Microbiology, from the teams of Lucy Shapiro, Martin Thanbichler and Christine Jacobs‐Wagner, describe a novel protein, called DipM for Division Involved Protein with LysM domains, that is required for cell division in Caulobacter crescentus. DipM localizes to the mid‐cell during cell division, where it is necessary for the hydrolysis of the septal peptidoglycan to remodel the cell wall. Loss of DipM results in severe defects in cell envelope constriction, which is deleterious under fast‐growth conditions. State‐of‐the‐art microscopy experiments reveal that the peptidoglycan is thicker and that the cell wall is incorrectly organized in DipM‐depleted cells compared with wild‐type cells, demonstrating that DipM is essential for reorganizing the cell wall at the division site, for envelope invagination and cell separation in Caulobacter.     

Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa. Received: 4 June 1999 / Accepted: 6 July 1999     

Expanding the paradigm for the outer membrane: Acinetobacter baumannii in the absence of endotoxin

Asymmetry in the outer membrane has long defined the cell envelope of Gram‐negative bacteria. This asymmetry, with lipopolysaccharide (LPS) or lipooligosaccharide (LOS) exclusively in the outer leaflet of the membrane, establishes an impermeable barrier that protects the cell from a number of stressors in the environment. Work done over the past 5 years has shown that Acinetobacter baumannii has the remarkable capability to survive with inactivated production of lipid A biosynthesis and the absence of LOS in its outer membrane. The implications of LOS‐deficient A. baumannii are far‐reaching – from impacts on cell envelope biogenesis and maintenance, bacterial physiology, antibiotic resistance and virulence. This review examines recent work that has contributed to our understanding of LOS‐deficiency and compares it to studies done on Neisseria meningitidis and Moraxella catarrhalis; the two other organisms with this capability.     

PapA,a peptidoglycan-associated protein,interacts with OmpC and maintains cell envelope integrity

The bacterial cell envelope is critical to support and maintain cellular life. In Gram-negative bacterial cells, the outer membrane and the peptidoglycan layer are two important parts of the cell envelope and they harbour abundant proteins. Here, we report the identification and characterization of a previously unknown p eptidoglycan-a ssociated p rotein, PapA, from the Gram-negative Comamonas testosteroni. PapA bound peptidoglycan with its C-terminal domain and interacted with the outer-membrane porin OmpC. The PapA-OmpC complex riveted the outer membrane and the peptidoglycan layer, and played a role in maintaining cell envelope integrity. When papA was disrupted, the mutant CNB-1ΔpapA apparently had an outer membrane partly separated from the peptidoglycan layer. Phenotypically, the mutant CNB-1ΔpapA lost chemotactic responses and had longer lag-phase of growth, less flagellation and higher sensitivity to harsh environments. Totally, 1093 functionally unknown PapA homologues were identified from the public NR protein database and they were mainly distributed in Burkholderiales of Betaproteobacteria. Our finding provides a clue that the PapA homologous proteins might function as a rivet to maintain cell envelope integrity in those Gram-negative bacteria.     

DipM links peptidoglycan remodelling to outer membrane organization in Caulobacter

Cell division in Gram‐negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). Here, we identify DipM, a putative LytM endopeptidase in Caulobacter crescentus, and show that it plays a critical role in maintaining cell envelope architecture during growth and division. DipM localized to the division site in an FtsZ‐dependent manner via its PG‐binding LysM domains. Although not essential for viability, ΔdipM cells exhibited gross morphological defects, including cell widening and filamentation, indicating a role in cell shape maintenance and division that we show requires its LytM domain. Strikingly, cells lacking DipM also showed OM blebbing at the division site, at cell poles and along the cell body. Cryo electron tomography of sacculi isolated from cells depleted of DipM revealed marked thickening of the PG as compared to wild type, which we hypothesize leads to loss of trans‐envelope contacts between components of the Tol–Pal complex. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Surface Display of the Receptor-Binding Region of the Lactobacillusbrevis S-Layer Protein in Lactococcuslactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein

The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.     

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.     

Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.     

The structure of Lactobacillus brevis surface layer reassembled on liposomes differs from native structure as revealed by SAXS

The reassembly of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 on positively charged liposomes was studied by small angle X-ray scattering (SAXS) and zeta potential measurements. SlpA was reassembled on unilamellar liposomes consisting of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-3-trimethylammonium-propane, prepared by extrusion through membranes with pore sizes of 50 nm and 100 nm. Similarly extruded samples without SlpA were used as a reference. The SlpA-containing samples showed clear diffraction peaks in their SAXS intensities. The lattice constants were calculated from the diffraction pattern and compared to those determined for SlpA on native cell wall fragments. Lattice constants for SlpA reassembled on liposomes (a = 9.29 nm, b = 8.03 nm, and γ = 84.9°) showed a marked change in the lattice constants b and γ when compared to those determined for SlpA on native cell wall fragments (a = 9.41 nm, b = 6.48 nm, and γ = 77.0°). The latter are in good agreement with values previously determined by electron microscopy. This indicates that the structure formed by SlpA is stable on the bacterial cell wall, but SlpA reassembles into a different structure on cationic liposomes. From the (10) reflection, the lower limit of crystallite size of SlpA on liposomes was determined to be 92 nm, corresponding to approximately ten aligned lattice planes.     

Annual fish oogenesis : II. Formation of the secondary egg envelope

Elaborate surface ornamentation of the secondary egg envelope of the annual fishes, Cynolebias melanotaenia and C. ladigesi, is comprised of 250-Å components which are synthesized in the follicle cells during Stage 5 and are secreted during Stage 6. Tubules, due to their structure and electron density, act as naturally occurring tracers which can be used to localize their site of synthesis and path of intracellular transport. At Stage 5, follicle cells acquire the morphological features of a protein synthesizing-secreting cell. Appearance of a well-developed granular endoplasmic reticulum coincides with the initial appearance of tubular material. Tubular precursors first appear in the cisternae of the RER. These regions become dilated and seem to bud off to form vesicles. Vesicle enlargement occurs through continued synthesis or by fusion with other vesicles. Vesicles contain partially assembled tubules in a loose aggregation of disks and short rods. The limiting membrane of vesicles, even up to half the size of the nucleus, is uniformly studded with ribosomes. Late in Stage 5, the contents of the vesicles coalesce into compact granules in which 250-Å tubules have assembled. During Stage 6, shortly before fusing with the plasma membrane, the RER-derived vesicles appear to shed their ribosomes. Upon fusion, the vesicle discharges a discrete granule. The RER-derived vesicles transport tubular components directly from the site of synthesis to the exterior of the cell. Morphological evidence indicates that the pathway of intracellular transport and secretion bypasses the Golgi complex. Hexagonal close packing of the follicle cells seems to determine the spatial distribution of pattern in the secondary envelope. Generation of elaborate structures within the overall pattern results from the morphogenetic activity of individual cells. On the basis of structure, the secondary envelope constitutes a chorionic respiratory system similar to insect eggs. It probably functions as a “plastron” or physical gill.