Novel primers for the amplification of nuclear DNA introns in the entomopathogenic nematode Heterorhabditis bacteriophora and their cross-amplification in seven other Heterorhabditis species.

We describe 24 novel primers that amplify intron regions in housekeeping and structural genes of Heterorhabditis bacteriophora. The cross-amplification potential of these primers in seven other Heterorhabditis species was determined. The results obtained showed interspecific nucleotide, length and splice site variability in the sequenced introns and for one gene, an intron gain was observed. These primers will be useful tools for studying population genetics, genetic diversity and intron DNA evolution within the genus Heterorhabditis and other genera of rhabditid nematodes.

Detection of changes occurring during recovery from the dauer stage in Heterorhabditis bacteriophora.

Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. When conditions are unfavourable for reproduction in H. bacteriophora, a long-lived, non-feeding, survival and dispersal stage, the dauer juvenile (DJ), is formed. This DJ stage is also adapted for host finding and infection. When it infects a suitable host, the DJ recovers and resumes growth and development. We describe a series of methods for improved detection of recovery in H. bacteriophora. We also describe some of the physiological changes that occur immediately after the onset of recovery in these nematodes as revealed using fluorescent nucleic acid binding SYTO dyes. Although recovery could be monitored using morphological changes, we found that observation of the uptake of fluorescent latex microspheres by recovering nematodes was a far more sensitive and efficient means of detecting recovery. SYTO dyes were also found to be useful indicators of recovery, binding to the pharyngeal glands and genital primordia as little as 3 h after the onset of recovery. The use of SYTO dyes also indicated that the pharyngeal glands produce large quantities of RNA following the onset of recovery, implying that these structures may produce proteins important in the infection and/or feeding process of H. bacteriophora.

Functional comparison of the NAD binding cleft of ADP-ribosylating toxins.

Although a common core structure forms the active site of ADP-ribosylating (ADPRT) toxins, the limited-sequence homology within this region suggests that different mechanisms are being used by toxins to perform their shared function. To explain differences in their mechanisms of NAD binding and hydrolysis, the functional interrelationship of residues predicted to perform similar functions in the beta3-strand of the NAD binding cleft of different ADPRT toxins was compared. Replacing Tyr54 in the A-subunit of diphtheria toxin (DTA) with a serine, its functional homologue in cholera toxin (CT), resulted in the loss of catalytic function but not NAD binding. The catalytic role of the aromatic portion of Tyr54 in the ADPRT reaction was confirmed by the ability of a Tyr54-to-phenylalanine DTA mutant to retain ADPRT activity. In reciprocal studies, positioning a tyrosine in the beta3-strand of the A1-subunit of CT (CTA1) caused both loss of function and altered structure. The restricted flexibility of the CTA1 active site relative to function became evident upon the loss of ADPRT activity when a conservative Val60-to-leucine mutation was performed. We conclude from our studies that DT and CT maintain a similar mechanism of NAD binding but differ in their mechanisms of NAD hydrolysis. The aromatic moiety at position 54 in DT is integral to NAD hydrolysis, while NAD hydrolysis in CT appears highly dependent on the precise positioning of specific residues within the beta3-strand of the active-site cleft.

Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S.

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa 388 and the non-ExoS-producing strain 388DeltaS. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388DeltaS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388DeltaS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P. aeruginosa infectious process by rendering cells nonfunctional.

Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region.

LuxR, the Vibrio fischeri luminescence gene (lux) activator, is the best-studied member of a family of bacterial transcription factors required for cell density-dependent expression of specific genes involved in associations with eukaryotic hosts. Neither LuxR nor any other LuxR homolog has been shown to bind DNA directly. We have purified the LuxR C-terminal transcriptional activator domain from extracts of recombinant Escherichia coli in which this polypeptide was expressed. The purified polypeptide by itself binds to lux regulatory DNA upstream of the lux box, a 20-bp palindrome that is required for LuxR activity in vivo, but it does not bind to the lux box. However, the LuxR C-terminal domain together with RNA polymerase protects a region including the lux box and the lux operon promoter from DNase I cleavage. There is very little protection of the lux operon promoter region from DNase I digestion in the presence of RNA polymerase alone. Apparently, there is a synergistic binding of the LuxR C-terminal domain and RNA polymerase to the promoter region. The upstream binding region for the purified polypeptide encompasses a binding site for cAMP receptor protein (CRP). Under some conditions, CRP binding can block the binding of the LuxR C-terminal domain to the upstream binding region, and it can also block the synergistic binding of the LuxR C-terminal domain and RNA polymerase to the lux box and luminescence gene promoter region. This description of DNA binding by the LuxR C-terminal domain should lead to an understanding of the molecular interactions of the LuxR family of transcriptional activators with regulatory DNA.

Evidence that GroEL, not sigma 32, is involved in transcriptional regulation of the Vibrio fischeri luminescence genes in Escherichia coli.

In Escherichia coli, transcription of the inducible Vibrio fischeri luminescence operon, luxICDABE, has been reported to require sigma 32, the product of rpoH. Consistent with previous studies, we report that an E. coli delta rpoH mutant, KY1601 containing luxICDABE and luxR, which codes for the activator of luxICDABE transcription on a plasmid (pJE202), was weakly luminescent. Transformation of this E. coli strain with a plasmid containing rpoH under the control of the tac promoter resulted in high levels of cellular luminescence. However, the level of expression of the pJE202 luxICDABE was also high in E. coli 1603, a delta rpoH mutant with a second-site mutation that resulted in sigma 32-independent overexpression of the groE operon. Apparently, sigma 32 is not directly required for the transcription of luxICDABE in E. coli but is required for sufficient expression of groE, which is in turn required for the transcription of luxICDABE. This conclusion is supported by the finding that E. coli groE mutants containing pJE202 were weakly luminescent. In the E. coli delta rpoH mutant KY1601, the sigma 32 requirement for the transcription of luxICDABE was partially compensated for by the addition of saturating concentrations of the inducer to the culture medium and largely compensated for when cells were transformed with a luxR overexpression vector. These data support the hypothesis that sigma 32 is not required for transcription of luxICDABE. Rather, it appears that the products of groE are required for the folding of LuxR into an active protein, like they are for the folding of several other proteins.

Characterization of membrane-associated and soluble states of SecA protein from wild-type and SecA51(TS) mutant strains of Escherichia coli.

The subcellular localization of SecA, a protein essential for the catalysis of general protein export, was studied to better understand its state(s) and function(s) within Escherichia coli cells. In a wild-type strain approximately half of the cellular SecA content was found to be associated with the inner membrane, while the remainder was soluble. Association of SecA protein with the inner membrane required the presence of anionic phospholipids and was modulated by ATP. A fraction of the membrane-bound SecA was found to be integrally associated with the membrane. In the secA51(Ts) mutant 75-95% of SecA protein was found to be membrane associated, independent of the protein export status of the cell, implying that the partitioning of this protein between the cell membrane and cytoplasm may play an important role in its function. secA-lacZ fusions were used to map a membrane association determinant to the amino-terminal quarter of SecA protein sequence. When this portion of SecA protein was expressed within cells, it was found solely in membrane fractions and complemented the growth and protein secretion defect of the secA51(Ts) mutant. This indicates that the membrane is the site of the limiting defect in this mutant and suggests that either SecA functions can be divided into at least two separable activities or that productive interaction between SecA and the amino-terminal fragment can occur in vivo.

Characterization of Escherichia coli SecA protein binding to a site on its mRNA involved in autoregulation.

In order to understand further the autogenous regulation of Escherichia coli secA translation, we have set up a purified system to study the binding of SecA protein to portions of its mRNA. Specific SecA protein-RNA binding was demonstrated by UV cross-linking, filter binding, and gel shift assays. Use of the filter binding assay allowed optimization of binding, which was influenced by Mg2+ and ATP concentrations, and a measurement of the affinity of this interaction. A nested series of RNAs lacking either 5' or 3' portions of geneX-secA sequences were used to localize the SecA protein binding site to sequences around the geneX-secA intergenic region. These studies imply that SecA protein directly regulates its own translation by a specific RNA binding activity that presumably blocks translational initiation.

The first gene in the Escherichia coli secA operon, gene X, encodes a nonessential secretory protein.

TnphoA insertions in the first gene of the Escherichia coli secA operon, gene X, were isolated and analyzed. Studies of the Gene X-PhoA fusion proteins showed that gene X encodes a secretory protein, since the fusion proteins possessed normal alkaline phosphatase activity and a substantial portion of this activity was found in the periplasm. In addition, the Gene X-PhoA fusion proteins were initially synthesized with a cleavable signal peptide. A gene X::TnphoA insertion was used to construct a strain containing a disrupted chromosomal copy of gene X. Analysis of this strain indicated that gene X is nonessential for cell growth and viability and does not appear to play an essential role in the process of protein export.

Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element.

The Escherichia coli secA gene, whose translation is responsive to the proficiency of protein export within the cell, is the second gene in a three-gene operon and is flanked by gene X and mutT. By using gene fusion and oligonucleotide-directed mutagenesis techniques, we have localized this translationally regulated site to a region at the end of gene X and the beginning of secA. This region has been shown to bind SecA protein in vitro. These studies open the way for a direct investigation of the mechanism of secA regulation and its coupling to the protein secretion capability of the cell.

Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery.

Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coli mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coli plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.

Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria.

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.