The Type IV Fimbrial Subunit Gene (fimA) of Dichelobacter nodosus Is Essential for Virulence, Protease Secretion, and Natural Competence

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.     

Plasmid ColE3 specifies a lysis protein.

Tn5 insertion mutations in plasmid ColE3 were isolated and characterized. Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium. Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells. All of these lysis mutants mapped near, but outside of, the structural genes for colicin E3 and immunity protein. Cells carrying the insertion mutations which did not release colicin E3 into the medium were not killed by UV exposure at levels that killed cells bearing wild-type plasmids. The protein specified by the lysis gene was identified in minicells and in mitomycin C-induced cells. A small protein, with a molecular weight between 6,000 and 7,000, was found in cells which released colicin into the medium, but not in mutant cells that did not release colicin. Two mutants with insertions within the structural gene for colicin E3 were also characterized. They produced no colicin activity, but both synthesized a peptide consistent with their map position near the middle of the colicin gene. These two insertion mutants were also phenotypically lysis mutants--they were not killed by UV doses lethal to wild-type colicinogenic cells and they did not synthesize the small putative lysis protein. Therefore, the lysis gene is probably in the same operon as the structural gene for colicin E3.     

Isolation and characterization of OmpC porin mutants with altered pore properties.

The LamB protein is normally required for the uptake of maltodextrins. Starting with a LamB- OmpF- strain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex+ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB- OmpF- strains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of [14C]maltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain beta-lactam antibiotics and sodium dodecyl sulfate, but did not exhibit an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.     

The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1----2)-glucan

The ndvB locus of Rhizobium meliloti was sequenced and found to encode a 319-kDa protein involved in the production of beta-(1----2)-glucan. Transposon Tn5 mutagenesis revealed that a large portion of the downstream half of this gene is not essential for symbiosis but is required for optimal production of beta-(1----2)-glucan. A high molecular weight inner membrane protein, believed to be the ndvB gene product, was absent from two different upstream ndvB::Tn5 mutants. This protein could be labeled in vitro with UDP-[U-14C]glucose in the wild type but not in the symbiotically defective mutants. Inner membrane preparations from the symbiotically competent downstream mutants labeled less well than did those from wild type with UDP-[U-14C] glucose and did not show distinct bands after polyacrylamide gel electrophoresis and fluorography, suggesting that C-terminal truncations of NdvB might affect the stability of this molecule. These downstream mutants had reduced amounts of periplasmic beta-(1----2)-glucan and exhibited several vegetative defects seen also in the upstream mutants. These included alterations in phage and antibiotic sensitivity, in motility, and in growth in low osmolarity media. Bacteroids produced by two of the downstream mutants were morphologically abnormal, indicating that ndvB is involved not only in invasion but also in bacteroid development.     

Transformation by v-myb correlates with trans-activation of gene expression.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.     

Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in Streptococcus pneumoniae

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.     

Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.     

Isolation and characterization of the yeast las21 mutants, which are sensitive to a local anestheticum, tetracaine.

We isolated and characterized yeast mutants whose growth is sensitive to a local anestheticum tetracaine and, at the same time, temperature sensitive. These mutants were collectively called las mutants (local anestheticum sensitive). The las21 mutants were analyzed in this study. The wild type LAS21 gene was cloned by exploiting temperature sensitivity of the las21 mutants and we found that LAS21 encodes ORF YJL062w which has not been analyzed before. Las21p is putative membrane protein belonging to the major facilitator super family containing plural membrane spanning domains. Complete elimination of the LAS21 ORF did not kill the cells but made their growth temperature sensitive. Interestingly, the complete loss of the LAS21 gene canceled the sensitivity to tetracaine. The ability of the las21 mutants to grow at a higher temperature was recovered in the various media containing an osmotic stabilizer or salts. Furthermore, temperature sensitivity of the las21 mutants was partially suppressed by introduction of PKC1, encoding protein kinase C, on a high copy vector. We found some genetic interactions between LAS21 and Ras/cAMP cascade genes. These results suggest that LAS21 defines unknown pathway regulating the stress response of yeast.     

Functional organization of the autotransporter adhesin involved in diffuse adherence

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. To elucidate the structure-function relationships of AIDA-I, we performed transposon-based linker scanning mutagenesis and constructed mutants with site-directed deletions. Twenty-nine different mutants with insertions that did not affect protein expression were obtained. Eleven mutants were deficient for one or two but not all of the functions associated with the expression of AIDA-I. Functional characterization of the transposon mutants and of an additional deletion mutant suggested that the N-terminal third of mature AIDA-I is involved in binding of this protein to cultured epithelial cells. The purified product of the putative domain could bind to cultured epithelial cells, confirming the importance of this region in adhesion. We also identified several different mutants in which invasion and adhesion were changed to different extents and two mutants in which autoaggregation and biofilm formation were also affected differently. These results suggest that although conceptually linked, adhesion and invasion, as well as autoaggregation and biofilm formation, are phenomena that may rely on distinct mechanisms when they are mediated by AIDA-I. This study sheds new light on the workings of a protein belonging to an emerging family of strikingly versatile virulence factors.     

Bacteriophage chi sensitivity and motility of Escherichia coli K-12 and Salmonella typhimurium Fla- mutants possessing the hook structure.

The production of hook protein and flagellin in 29 Fla- mutants of Escherichia coli K-12 was determined by the complement fixation assay. Six mutants produced hook protein, and four of them also produced flagellin. A flaE mutation was introduced into these fla mutants carrying the hook structure. All of these mutants made polyhooks and were used as hosts for a newly isolated host-range mutant of chi phage that has a high affinity for the hook structure. All except one mutant produced significant amounts of progeny phages. A flaD flaE double mutant was that exception which did not yield significant amounts of progeny by the phage propagation method. All of the flaE double mutants produced comparable amounts of polyhooks, and no qualitative difference was detected between chi-sensitive and chi-insensitive mutants by the complement fixation assay. Accordingly, it was thought that the polyhook of the flaD flaE mutant had a mechanical defect for chi phage infection. This assumption was confirmed by tethered-cell experiments; the flaD flaE mutant did not rotate. These results are well explained by a proposed regulation pathway of flagellar genes. flaE mutants can express other genes which govern the final step of the flagellar morphogenesis, whereas flaD mutants cannot rotate, possibly because the mocha operon is not expressed. The results obtained in E. coli were also found to be applicable to Salmonella typhimurium.     

Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage.

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein.     

Cyclic AMP and its receptor protein are required for expression of transfer genes of conjugative plasmid F in Escherichia coli

A number of cya and crp mutants of Escherichia coli HfrH were analyzed for several Tra functions of the F plasmid. The mutants were observed to be deficient in conjugal donor ability, absorption of phages MS2 and Q beta and surface exclusion. These defects were suppressed in cya mutants grown with cAMP supplementation. A cAMP concentration of 3 X 10(-4) M produced maximal suppression of donor ability defect in a cya strain. cAMP did not suppress the Tra- phenotype of crp mutants. Latent periods of MS2 were shorter in cya and crp bacteria. Phage T7 development appeared similar in wild type, cya, and crp cells. It is concluded that tra genes of F plasmid are expressed only to a small extent in cya and crp mutants and that cAMP and its receptor protein are required for the normal expression of tra genes.     

pH-dependent photoautotrophic growth of specific photosystem II mutants lacking lumenal extrinsic polypeptides in Synechocystis PCC 6803

The removal of either the PsbU or PsbV protein has been investigated in a cyanobacterial DeltaPsbO strain and in mutants carrying deletions or substitutions in lumen-exposed domains of CP47. These experiments have demonstrated a functional interaction between the PsbU protein and photosystem II (PSII) in the absence of the PsbO subunit. The control:DeltaPsbO:DeltaPsbU strain assembled PSII centers at pH 7.5 but did not evolve oxygen; however, photoautotrophic growth was restored at pH 10.0. In addition, several CP47 mutants, lacking extrinsic proteins, were obligate photoheterotrophs at pH 7.5 but photoautotrophic at pH 10.0, whereas other strains remained photoheterotrophs at alkaline pH.     

A unique beta-hairpin protruding from AAA+ ATPase domain of RuvB motor protein is involved in the interaction with RuvA DNA recognition protein for branch migration of Holliday junctions.

The Escherichia coli RuvB protein is a motor protein that forms a complex with RuvA and promotes branch migration of Holliday junctions during homologous recombination. This study describes the characteristics of two RuvB mutants, I148T and I150T, that do not promote branch migration in the presence of RuvA. These RuvB mutants hydrolyzed ATP and bound duplex DNA with the same efficiency as wild-type RuvB, but the mutants did not form a complex with RuvA and were defective in loading onto junction DNA in a RuvA-assisted manner. A recent crystallographic study revealed that Ile(148) and Ile(150) are in a unique beta-hairpin that protrudes from the AAA(+) ATPase domain of RuvB. We propose that this beta-hairpin interacts with hydrophobic residues in the mobile third domain of RuvA and that this interaction is vital for the RuvA-assisted loading of RuvB onto Holliday junction DNA.     

Inhibitory effect of myristylation on transrepression by FBR (Gag-Fos) protein.

The myristylated v-fos product, FBR murine sarcoma virus (Gag-Fos) protein, exhibits a lower level of transrepression of the serum response element (SRE) than does c-fos protein (Fos). Mutation of the N-terminal myristylation site in FBR protein restored SRE transrepression. Replacement of N-terminal viral Gag sequences with the Fos N terminus also restored this activity, providing additional evidence that myristylation inhibits transrepression by FBR protein. However, the myristylated Gag domain did not inhibit SRE transrepression when fused to Fos, indicating that myristylation of a fos protein is not by itself sufficient to prevent SRE transrepression and that C-terminal mutation is necessary to inhibit transrepression by N myristylation. Comparison of transfection results with Fos C-terminal deletion mutants and the Fos/FBR chimeric mutant revealed that the FBR C terminus retained the potential for transrepression despite deletion of the normal Fos C terminus, whereas similar Fos deletion mutants did not. These results indicate that both N- and C-terminal mutations are required to inhibit transrepression by FBR protein and that multiple structural mutations accompanied by posttranslational protein modification alter gene regulation by FBR protein.     

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein

We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protein 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8 gene so that an ICP8-beta-galactosidase fusion protein was expressed. This virus formed blue plaques on ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Mutated ICP8 gene plasmids cotransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinants were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed ICP8 that neither bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen.