NopB, a soybean cultivar-specificity protein from Sinorhizobium fredii USDA257, is a type III secreted protein

The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NoIB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.     

A nopA Deletion Mutant of Sinorhizobium fredii USDA257, a Soybean Symbiont, is Impaired in Nodulation

Sinorhizobium fredii USDA257 employs type III secretion system (T3SS) to deliver effector proteins into the host cells through pili. The nopA protein is the major component of USDA257 pili. The promoter region of USDA257 nopA possesses a well conserved tts box. Serial deletion analysis revealed that the tts box is absolutely essential for flavonoid induction of nopA. Deletion of nopA drastically lowered the number of nodules formed by USDA257 on cowpea and soybean cultivar Peking. In contrast to the parental strain, the USDA257 nopA mutant was able to form few nodules on soybean cultivars McCall and Williams 82. Light and transmission electron microscopy examination of these nodules revealed numerous starch grains both in the infected and uninfected cells.     

Y4xP, an open reading frame located in a type III protein secretion system locus of Sinorhizobium fredii USDA257 and USDA191, encodes cysteine synthase

Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production.     

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.     

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.     

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85

β‐barrel‐shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell‐wall of Gram‐negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane‐localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N‐terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline‐rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.     

Sinorhizobium fredii USDA257, a cultivar-specific soybean symbiont, carries two copies of y4yA and y4yB, two open reading frames that are located in a region that encodes the type III protein secretion system

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean (Glycine max) cultivar Peking but fails to nodulate the improved cultivar McCall. Cultivar specificity is governed by a plasmid-borne locus, nolXBTUV. By DNA sequence analysis, we have identified two open reading frames, y4yA and y4yB, immediately downstream of nolX. Northern (RNA) blot analysis indicated that the expression of both y4yA and y4yB is inducible by isoflavonoids, and an intact copy of nolX is required. Two copies each of y4yA and y4yB are present in S. fredii USDA257, one on the sym plasmid (y4yAsp and y4yBsp), and the other on the chromosome (y4yAc and y4yBc). The cultivar-nonspecific strain USDA191 lacks y4yAc and y4yBc. Introduction of y4yAc plus y4yBc from USDA257 into USDA191 did not influence the ability of the latter strain to nodulate McCall soybean plants. Unlike nolX, the inactivation of y4yAsp and y4yBsp of USDA257 did not extend the host range of this strain. A double mutant, in which both the plasmid and chromosomal copies of y4yA and y4yB were mutated, had no observable effect on symbiotic ability of USDA257. The y4yAsp and y4yBsp mutants did not influence flavonoid-dependent extracellular protein production. Rhizobium sp. strain NGR234 and S. saheli USDA4893 both contain sequences similar to S. fredii USDA257 y4yAsp and y4yBsp; however, Bradyrhizobium spp., the traditional soybean symbionts, lack these genes.     

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) cleaves and releases a 22-kDa extracellular matrix metalloproteinase inducer (EMMPRIN) fragment from tumor cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.     

Serological diagnostic potential of recombinant outer membrane protein (Omp31) from Brucella melitensis in goat and sheep brucellosis

Outer membrane proteins of Brucella have been classified as group 1 (94 or 88 kDa), group 2 (36–38 kDa), and group 3 (31–34 and 25–27 kDa). Two proteins of 25 and 31 kDa with only 34% of identity are included in group 3 and they are coded for by the omp25 and omp31 genes. Proposed study planned to detect antibodies to Brucella melitensis Omp31 in farm goats having history of B. melitensis induced abortions, in B. melitensis-infected goats and sheep. By enzyme-linked immunosorbent assay (ELISA), using recombinant Omp31 as antigen, of 872 farm goats antibodies to Omp31 were detected in 112 (12.8%) cases. Out of 14 naturally infected goats infected with B. melitensis 12 (85.7%) showed anti Omp31 antibodies. Out of 10 naturally infected sheep with Brucella ovis, antibodies to Omp31 were detected only in 6 (60%) cases and in 18 (81.8%) out of 22 cases infected with B. melitensis. Obtained results were also compared with the rose Bengal plate test (RBPT). In controlled experiments, sensitivity and specificity of recombinant Omp31 (rOmp31) ELISA and RBPT were also evaluated and it was found that former test is 100% specific though RBPT has slightly higher sensitivity. In this study, we found a significant difference between the two groups (B. melitensis and B. ovis infected) in terms of the percentage of positive reactions or signal level by an ELISA. The reactivity of the positive sera against the purified rOmp31 was also tested by Western blotting. Sera from B. melitensis-infected animals showed a strong reactivity in comparison to sera from B. ovis-infected animals. The potential diagnostic usefulness of this antigen in combination with other recombinant proteins from B. melitensis would be of great importance in future in eradication of brucellosis.     

Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family

Proteins of the Omp85 family chaperone the membrane insertion of β‐barrel‐shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear‐encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N‐terminal polypeptide transport‐associated (POTRA) domains and a C‐terminal membrane‐embedded β‐barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β‐barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75‐V, which is consistent with the phylogenetic clustering of P39 in the Toc75‐V rather than the Toc75‐III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75‐III, Toc75‐V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391–1401. © 2014 Wiley Periodicals, Inc.     

YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli

The Omp85 family of proteins has been found in all Gram-negative bacteria and even several eukaryotic organisms. The previously uncharacterized Escherichia coli member of this family is YaeT. The results of this study, consistent with previous Omp85 studies, show that the yaeT gene encodes for an essential cellular function. Direct examinations of the outer membrane fraction and protein assembly revealed that cells depleted for YaeT are severely defective in the biogenesis of outer membrane proteins (OMPs). Interestingly, assemblies of the two distinct groups of OMPs that follow either SurA- and lipopolysaccharide-dependent (OmpF/C) or -independent (TolC) folding pathways were affected, suggesting that YaeT may act as a general OMP assembly factor. Depletion of cells for YaeT led to the accumulation of OMPs in the fraction enriched for periplasm, thus indicating that YaeT facilitates the insertion of soluble assembly intermediates from the periplasm to the outer membrane. Our data suggest that YaeT's role in the assembly of OMPs is not mediated through a role in lipid biogenesis, as debated for Omp85 in Neisseria, thus advocating a conserved OMP assembly function of Omp85 homologues.     

Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia

Abstract A 66-kDa protein (p66) associated with the outer membrane of Lyme disease Borrelia was analysed at the molecular level. The chromosomal genes encoding p66 in B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90 were sequenced. Database searches revealed that the p66 gene sequences were homologous to a previously reported gene fragment of unknown function. The deduced amino acid sequences of p66 in different Lyme disease borreliae were 92–94% identical and had no homologs in the databases. Proteolytic cleavage patterns of p66 and a computer-predicted single trans-membrane helix suggested the presence of surface-exposed epitopes on the C-terminus.     

Cloning of the locus encoding synthesis of an outer membrane protein (Omp2) of the calcium dependence plasmid of Yersinia pestis (Lehmann, Neumann)

The genetic locus of Yersinia pestis encoding synthesis of a 46-kDa heat-inducible outer membrane protein (Omp2) was cloned into pBR322 plasmid. The Omp2 was shown to be analogous to previously described YopH and Yop2b proteins. The fifth HindIII fragment of 48-MDa calcium dependence plasmid pCad358 mediates production of 31- and 28-kDa proteins, irrespective of orientation of the insertion. A 31-kDa polypeptide seems to correspond to the YopJ described elsewhere. The maps of BamHI and HindIII of pCad358 region studied differed from those described for pCD1 plasmid of Y. pestis KIM. The products encoded by genes from the fragment cloned in the Pgm+ background give rise to considerable growth of Y. pestis within mouse peritoneal macrophages but were not sufficient to cause lethal infectious process.     

Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.     

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.     

Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria

As a consequence of their bacterial origin, mitochondria contain β-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the β-barrel assembly machinery (BAM) required for biogenesis of β-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial β-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only β-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial β-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of β-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of β-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.     

MOMP (major outer membrane protein) of Campylobacter jejuni; a versatile pore-forming protein

The great majority of trimeric porins of Gram-negative bacteria cannot be dissociated into monomers without disrupting their folded conformation. The porin of Campylobacter jejuni, however, displays two folded structures, a classical oligomer and a monomer resistant to detergent denaturation. We probed the transition of trimer to monomer using light scattering experiments and examined the secondary structures of these two molecular states by infra-red spectroscopy. The channel-forming properties of both trimer and monomer were studied after incorporation into artificial lipid bilayers. In these conditions, the trimer induced ion channels with a conductance value of 1200 pS in 1 M NaCl. The pores showed marked cationic selectivity and sensitivity to low voltage. Analysis of the isolated monomer showed nearly the same single-channel conductance and the same selectivity and sensitivity to voltage. These results indicate that the folded monomer form of C. jejuni MOMP displays essentially the same pore-forming properties as the native trimer.     

The Acinetobacter outer membrane protein A (OmpA) is a secreted emulsifier

Acinetobacter strains use hydrophobic carbon sources and most of them are efficient oil degraders. They secrete a variety of emulsifiers which are efficient in producing and stabilizing oil-in-water emulsions. The bioemulsifier of Acinetobacter radioresistens KA53 (Alasan) is a high-mass complex of proteins and polysaccharides. The major emulsification activity of this complex is associated with a 45 kDa protein (AlnA), which is homologous to the outer membrane protein OmpA. The emulsification ability of AlnA depends on the presence of hydrophobic residues in the four loops spanning the transmembrane domains. The finding of a secreted OmpA was unexpected, in view of the fact that this protein is essential in all Gram-negative bacteria, has four trans-membrane domains and is considered to be an integral structural component of the outer membrane. However, secretion of an OmpA with emulsifying ability could be of physiological importance in the utilization of hydrophobic substrates as carbon sources. Here we examined the possibility that secretion of OmpA with emulsifying activity is a general property of the oil-degrading Acinetobacter strains. The results indicate that OmpA is secreted in five strains of Acinetobacter, including strain Acinetobacter sp. ADP1 whose genome has been sequenced. The ompA genes of ADP1 and an additional strain, Acinetobacter sp. V-26 were cloned and sequenced. Structure analysis of the sequence of the two proteins indicated the existence of the hydrophobic regions, previously shown to be responsible for the emulsification activity of AlnA. Further examination of the recombinant OmpA proteins indicated that they are, indeed, strong emulsifiers, even when produced in Escherichia coli. The finding that Acinetobacter OmpA has emulsifying activity and that it is secreted in five strains of Acinetobacter may be physiologically significant and suggests the involvement of this protein in biodegradation of hydrophobic substrates, including hydrocarbons.