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Flavobacteria (members of the family Flavobacteriaceae ) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis , is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis , including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis . A flavobacterial expression system based on the promoter P ompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi , A. gambiae , and Aedes triseriatus , respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis . Animal erythrocytes promoted E. anophelis growth in vivo and in vitro , indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion ( eps ) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae . Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC , the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface. IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59 A resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160–166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104–135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.

The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila , GspA Ah , is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB Ah ). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD Ah ) secretin multimer in the outer membrane. In this study, the requirement for GspAB in the assembly of the T2SS secretin in Aeromonas and Vibrio species was investigated. We have demonstrated a requirement for GspAB in T2SS assembly in Aeromonas salmonicida , similar to that previously observed in A. hydrophila . In the Vibrionaceae species Vibrio cholerae , Vibrio vulnificus , and Vibrio parahaemolyticus , gspA mutations significantly decreased assembly of the secretin multimer but had minimal effects on the secretion of T2SS substrates. The lack of effect on secretion of the mutant of gspA of V. cholerae ( gspA Vc ) was explained by the finding that native secretin expression greatly exceeds the level needed for efficient secretion in V. cholerae . In cross-complementation experiments, secretin assembly and secretion in an A. hydrophila gspA mutant were partially restored by the expression of GspAB from V. cholerae in trans , further suggesting that GspAB Vc performs the same role in Vibrio species as GspAB Ah does in the aeromonads. These results indicate that the GspAB complex is functional in the assembly of the secretin in Vibrio species but that a redundancy of GspAB function may exist in this genus.

Summary The type II secretion system is a multi-protein complex that spans the cell envelope of gramnegative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsGK, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189: 8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

We investigated the bacterial community composition of tree holes in relation to the presence and absence of larvae of the mosquito Ochlerotatus triseriatus. Larvae were eliminated from a subset of natural tree holes with Bacillus thuringiensis serovar israelensis, and total bacterial numbers, slow- and fast-growing colony-forming units on minimal media, and 16S rRNA gene sequence data from water column and leaf material were obtained. Total bacterial counts did not change significantly with treatment; however, the number of slow-growing cultivable bacteria significantly increased in the absence of larvae. Sequence classifications and comparisons of sequence libraries using LIBSHUFF indicated that the elimination of larvae significantly altered bacterial community composition. Major groups apparently affected by larvae were Flavobacteriaceae, Rhodobacteraceae, Comamonadaceae, and Sphingomonadaceae. A clear dominance of Flavobacteriaceae in the water column after larval removal suggests members of this group are a major bacterial food source.

A cluster of six genes from Flavobacterium hibernum strain W22, fus-rpsJ-rplC-rplD-rplW-rplB, was cloned and sequenced. A short fragment upstream of rplC, but not of rpsJ, showed strong promoter activity in flavobacteria. TATCTTTG and TTG motifs that are conserved in Flavobacterium promoters were found immediately upstream of the transcription start point of rplC, at the -7 and -33 positions, respectively. RT-PCR analysis of the transcripts revealed that rpsJ and rplC are expressed as separate transcriptional units, whereas rplC and rplD-rplW-rplB are cotranscribed as a single mRNA, in contrast to the situation in the Gammaproteobacteria, which have a single transcriptional unit of 11 ribosomal S10 genes.

The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.

The type II secretion system (T2SS) is used by several Gram-negative bacteria for the secretion of hydrolytic enzymes and virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 so-called "Gsp proteins" spans from a regulatory ATPase in the cytoplasm, via several signal or energy transducing proteins in the inner membrane and the pseudopilins in the periplasm, to the actual pore in the outer membrane. The human pathogen Vibrio cholerae employs such an assembly, called the Eps system, for the export of its major virulence factor, cholera toxin, from its periplasm into the lumen of the gastro-intestinal tract of the host. Here, we report the atomic structure of the major cytoplasmic domain of the inner membrane-spanning EpsL protein from V. cholerae. EpsL is the binding partner of the regulatory ATPase EpsE as well as of EpsM and pseudopilins, and is therefore a critical link between the cytoplasmic and the periplasmic part of the Eps-system. The 2.7A resolution structure was determined by a combination of Se-Met multiple anomalous dispersion (MAD) and multiple isomorphous replacement with anomalous scattering (MIRAS) phasing methods. The 28kDa cytoplasmic domain of EpsL (cyto-EpsL) consists of three beta-sheet-rich domains. With domains I and III similar to the RNaseH-fold, cyto-EpsL unexpectedly shows structural homology with the superfamily of actin-like ATPases. cyto-EpsL, however, is an unusual member of this superfamily as it misses the canonical actin domains 1B and 2B, which are common yet variable in this superfamily. Moreover, cyto-EpsL has an additional domain II, which has the topology of an SHS2-fold module. Within the superfamily this fold module has been observed only for domain 1C of the cell division protein FtsA, in which it mediates protein-protein interactions. This domain II displays great flexibility and contributes to a pronounced negatively charged canyon on the surface of cyto-EpsL. Functional data as well as structural homology and sequence conservation suggest that domain II interacts with EpsE, the major cytoplasmic binding partner of EpsL.

A Shigella dysenteriae 1 strain isolated from an epidemic in West Bengal, India. The strain contained six plasmids including a large virulence plasmid. A plasmid, pPR1347 carrying both the rfb gene cluster and the rfc gene of Salmonella typhimurium has been transferred to this invasive Shigella dysenteriae 1 strain by triparental cross with a very low frequency. Only five stable (100%) clones were isolated after examining several thousand colonies. All five transconjugants were Sereny negative and were unable to invade the HeLa cells. Transconjugants exhibited strong cross reactivity with S. dysenteriae 1 antisera but they showed weak reaction with Salmonella typhimurium antisera. Plasmid profiles of the transconjugants were unaltered as compared with the wild type strain except for the presence of pPR 1347. The transconjugants regained their invasive property after elimination (curing) of pPR 1347. However, Shigella-infected convalescent phase serum was able to detect IpaABCD proteins from whole cell lysate and culture supernatant of transconjugants and cured (pPR 1347) transconjugants. A 60 kDa IpaH protein was not secreted into the culture supernatant by the transconjugants. Synthesis of lipopolysaccharides (LPS) of the hybrid strains was increased within the region of 43 to 67 kDa in comparison with the wild type S. dysenteriae 1 strains.

In vitro gene manipulation was used to develop a novel chimeric antigen consisting of the non-toxic B subunit (EtxB) of an E. coli enterotoxin and the first 14 N-terminal amino acid residues of the carboxy-terminal portion of the α subunit of bovine inhibin (bINH 1–14 ). Rabbits immunized subcutaneously (s.c.) or intravenously (i.v.) with EtxB::bINH 1–14 , with or without Freund's adjuvant, developed significant titres of antibodies that recognized an inhibin peptide fragment containing bINH 1–14 , native inhibins, and EtxB during separate enzyme-linked immunosorbent assay (ELISA). Passive immunization of mice with the rabbit anti-EtxB::bINH 1–14 serum increased concentrations of follicle-stimulating hormone (FSH) in serum twofold compared with controls, whereas serum concentrations of luteinizing hormone (LH) were unaltered. Since FSH is the primary hormone from the pituitary gland that stimulates ovarian follicle growth and spermatogenesis, the results of this study demonstrate that EtxB::bINH 1–14 has potential as antigen for development of inhibin-based fertility vaccines.

During the past few years, significant progress has been made towards our understanding of the molecular mechanisms governing the translocation of proteins through bacterial cell membranes. Successful attempts in promoting the secretion of recombinant proteins by employing this knowledge and by empirical efforts have been registered. However, a further in-depth understanding of membrane-translocation mechanisms is required before predictable manipulations of secretion systems can be made to secrete native recombinant proteins that are not naturally targeted to the extracellular compartment.

The B-subunit pentamer(s) (EtxBp) of Escherichia coli heat-labile enterotoxin (LT) are secreted from Vibrio cholerae via the general secretion pathway (GSP), but remain periplasmic in E. coli. In order to determine if other Gram- bacteria were also able to secrete the ExtBp, the etxB gene, which encodes EtxB was introduced into different bacteria. Of the bacteria examined, most species of Vibrio and Aeromonas were able to secrete this protein through the outer membrane; other Gram- genera, including Erwinia, Klebsiella and Xanthomonas were not, even though they encode GSP genes homologous to those of V. cholerae. Thus, the ability to recognize the EtxBp as a secretable protein is confined to bacteria that were identified as being closely related to V. cholerae by examination of their 5S rRNA [MacDonell and Colwell, Syst. Appl. Microbiol. 6 (1985) 171-182].

A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30 °C, but Xyn10A retained 50% activity at 4 °C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-β-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose.

The type II secretion system is a multi-protein complex that spans the cell envelope of gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B subunit (EtxB) affect its assembly into pentamers in a temperature-dependent manner. At 42 degrees C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30 degrees C assemble, in the periplasm, into pentamers that remain stable when transferred to 42 degrees C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65 degrees C. This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus. The Thr-->Ile replacement at position 75 in the alpha 2-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met-->Leu substitution at position 101 in the beta 6-strand of EtxB191.5. Interaction between the alpha 2-helix and beta 6-strand which contains the C-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.

The thermostability of D-xylose isomerase from Thermoanaerobacterium thermosulfurigenes was enhanced by site-directed substitutions of aromatic amino acids in the active site. This enhancement may be explained as the consequence of the reduction of the area of water-accessible hydrophobic surface. The kinetics of thermoinactivation of the enzyme in aqueous solution was also investigated, and we report that in addition to the well known divalent cations, the monovalent cation, K+, also protects the enzyme against thermoinactivation. The kinetic data suggest that the formation of incorrect conformations of the enzyme (“scrambled structure”) is the dominant factor governing the process of thermoinactivation at elevated temperature (80–90°C).

The functions of individual amino acid residues in the active site of Thermoanaerobacterium thermosulfurigenes D-xylose ketol-isomerase (EC 5.3.1.5) were studied by site-directed substitution. The role of aromatic residues in the active-site pocket was not limited to the creation of a hydrophobic environment. For example, Trp-188 provided for substrate binding and Trp-139 allowed for the discrimination between D-xylose and D-glucose. Substrate discrimination was accomplished by steric hindrance caused by the side chain of Trp-139 toward the larger glucose molecule. Preference of the enzyme for the alpha-anomer of glucose depended on the His-101/Asp-104 pair. Wide differences observed in the catalytic constant (kcat) for alpha- versus beta-glucose in the wild-type enzyme and the fact that only the kcat for alpha-glucose was changed in the His-101-->Asn mutants strongly suggest that the substrate molecule entering the hydride-shift step is still in the cyclic form. On the basis of these results a revised hypothesis for the catalytic mechanism of D-xylose isomerase has been proposed that involves His-101, Asp-104, and Asp-339 functioning as a catalytic triad.

Cholera toxin and the related heat-labile enterotoxin (LT) produced by Escherichia coli consist of a holotoxin of one A subunit and five B subunits (AB5). Here we investigate the domains of the A subunit (EtxA) of E. coli LT which influence the events of B-subunit (EtxB) oligomerization and the formation of a stable AB5 holotoxin complex. We show that the C-terminal 14 amino acids of the A subunit comprise two functional domains that differentially affect oligomerization and holotoxin stability. Deletion of the last 14 amino acids (-14) from the A subunit resulted in a molecule that was significantly impaired in its capacity to promote the assembly of a mutant B subunit, EtxB191.5. In contrast, deletion of the last four amino acids (-4) from the A subunit gave a molecule that retained such a capacity. This suggests that C-terminal residues within the -14 to -4 region of the A subunit are important for promoting the oligomerization of EtxB. In addition, we demonstrate that the truncated A subunit lacking the last 4 amino acids was unable to form a stable AB5 holotoxin complex even though it promoted B-subunit oligomerization. This suggests that the last 4 residues of the A subunit function as an "anchoring" sequence responsible for maintaining the stability of A/B subunit interaction during holotoxin assembly. These data represent an important example of how intermolecular interactions between polypeptides in vivo can modulate the folding and assembly of a macromolecular complex.

Summary PsiB, an anti-SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence-deduced molecular mass of 15741 daltons. Using an immuno-assay with anti-PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6-5 piasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn 10 outgoing promoter located upstream of psiB. When we inserted a Tn 10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6-5 PsiB. We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti-SOS function. We observed that PsiB protein was transiently expressed by a wild-type F sex factor during its transmission to an Escherichia coli K-12 recipient, In an F+× F- cross, PsiB concentration increased at least 10-fold in F- recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasm id replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single-stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single-stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory pheno-type.

Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication. Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative). Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC. When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase). The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained. It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.

Strong promoters were isolated from Flavobacterium johnsoniae in a promoter-trap vector incorporating a gfp reporter system, and were used to express fluorescent protein markers (including GFP, YFP, mOrange and mStrawberry) and insecticidal protein genes in Flavobacterium strains. Sequence analysis of trapped DNA fragments showed conserved Bacteroidetes promoter motifs (TTG-N 19 -TAnnTTTG) located upstream of putative open reading frames. Plasmids harboring these genomic DNA fragments from F. johnsoniae promoted strong production of fluorescent proteins in Flavobacterium hibernum but not in Escherichia coli . The most potent promoter (P ompA ) identified in this work was cloned upstream of genes encoding fluorescent proteins, and these were co-expressed in Flavobacterium strains. The p42 and p51 genes (binary toxins from Bacillus sphaericus ) when translationally fused to the 3′-end of gfp showed strong expression. Flavobacteria expressing these genes exhibited toxicity against larvae of the mosquitoes Culex quinquefasciatus , Anopheles gambiae , and Ochlerotatus triseriatus . However, transformants with the transcriptional fusion construct between cry11A with p20 from Bacillus thuringiensis did not express Cry11A protein indicating that constitutive expression of cry11A may be problematic in Flavobacterium .

A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.

Minimal alterations at the carboxyl terminus of the B subunit (EtxB) of heat-labile enterotoxin from Escherichia coli were found to have a marked effect on the assembly and release of this polypeptide into the periplasm. Nine mutant EtxB polypeptides were obtained by genetic manipulation of the 3'-end of the etxB gene using Bal31 nuclease digestion and codon substitution. A correlation was observed between the magnitude of the changes introduced at the carboxyl terminus and the extent to which the mutant polypeptides were defective in assembly and release. Some of the mutant B subunits, exemplified by those in which the last 2 amino acids had been deleted or in which the last 4 residues had been replaced by three different ones, were found to be only partially defective, with a proportion being associated with the periplasmic face of the cytoplasmic membrane and the remainder being exported to the periplasm. The portion associated with membranes was detected as monomers on sodium dodecyl sulfate-polyacrylamide gels, whereas the portion exported to the periplasm were detected as assembled oligomers. We conclude that the last few amino acids at the carboxyl terminus of EtxB exert a profound influence on the assembly and release of the B subunit from the cytoplasmic membrane during export in E. coli.

Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes , but not of proteobacteria, and they function poorly in Escherichia coli . In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σ ABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ 70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae .

We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (∼1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli , thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac , functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5′ rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.

Purified 2,4-dichlorophenoxyacetic acid (2,4-D)/(alpha)-ketoglutarate dioxygenase (TfdA) was shown to use 4-nitrophenoxyacetic acid (K(infm) = 0.89 (plusmn) 0.04 mM, k(infcat) [catalytic constant] = 540 (plusmn) 10 min(sup-1)), producing intensely yellow 4-nitrophenol. This reagent was used to develop a rapid, continuous, colorimetric assay for the detection of TfdA and analogous activities in 2,4-D-degrading bacterial cells and extracts.

Gram-negative bacteria use type II secretion systems for the transport of virulence factors and hydrolytic enzymes through the outer membrane. These sophisticated multi-protein complexes reach from the pore in the outer membrane via the pseudopilins in the periplasm and a multi-protein inner-membrane sub-complex, to an ATPase in the cytoplasm. The human pathogen Vibrio cholerae uses such a secretion machinery, called the Eps-system, for the export of its major virulence factor cholera toxin into the intestinal tract of the human host. Here, we describe the 2.4 A structure of the hetero-tetrameric complex of the N-terminal domain of the ATPase EpsE and the cytoplasmic domain of the inner membrane protein EpsL, which constitute the major cytoplasmic components of the Eps-system. A stable fragment of EpsE in complex with the cytoplasmic domain of EpsL was identified via limited proteolysis and facilitated the crystallization of the complex. This first structure of a complex between two different proteins of the type II secretion system reveals that the N-terminal domain of EpsE and the cytoplasmic domain of EpsL form a hetero-tetramer, in which EpsL is the central dimer and EpsE binds on the periphery. The dimer of EpsL in this complex is very similar to the dimer seen in the crystal structure of the native cytoplasmic domain of EpsL, suggesting a possible physiological relevance despite a relatively small 675 A2 buried solvent accessible surface. The N-terminal domain of EpsE, which forms a compact domain with an alpha+beta-fold, places its helix alpha2 in a mostly hydrophobic cleft between domains II and III of EpsL burying 1700 A2 solvent accessible surface. This extensive interface involves several residues whose hydrophobic or charged nature is well conserved and is therefore likely to be of general importance in type II secretion systems.

The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol. The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host. Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V.cholerae, which is a part of the interface between the regulating part and the rest of the Eps system. The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7A resolution. The monomer consists of two alphabetabeta-subdomains forming a sandwich of two alpha-helices and a four-stranded antiparallel beta-sheet. In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers. This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner. Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.

Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10 4 to 10 5 cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli . A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His 6 ) on a nickel chelating column, whereas Cry11A produced without the P20(His 6 ) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.

The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM of Vibrio cholerae , have been purified and characterized. Based on gel filtration analysis, both purified EpsM (His) 6 and wild-type EpsL present in an Escherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli , they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

We have cloned and characterized novel oxygenolytic ortho -dehalogenation ( ohb ) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISP OHB structural genes ohbA and ohbB , which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho -halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISP OHB utilized electron transfer components provided by the heterologous hosts. ISP OHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene ( ohbR ) was located upstream of the ohbAB genes. An open reading frame ( ohbC ) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli . The ohb genes are flanked by an IS 1396 -like sequence containing a putative gene for a 39,715-Da transposase A ( tnpA ) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III ( top ) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.

Peptides KDYGASTGSSL (Epi1), SLLRRRRNGEDVSV (Epi3) and DDEIYGNDGHP (Epi6), predicted to constitute immunogenic epitopes of the hemolysin-cytotoxin ApxIA of Actinobacillus pleuropneumoniae were inserted into a surface-exposed loop of the B subunit of the E. coli heat-labile enterotoxin (EtxB). The resulting chimeric proteins were recognized by monospecific antibodies against purified native ApxI and by convalescent sera of pigs that were positive for A. pleuropneumoniae serotype 1. Mice anti-sera against chimeric proteins EtxB::ApxIAEpi3 and EtxB::ApxIAEpi6 reacted with purified ApxI. These results indicate that Epi3 and Epi6 regions constitute linear epitopes of the structural ApxIA protein toxin. Epitope Epi6 which is located in the structure of the glycine rich repeats in ApxI elicits the formation of hemolysin neutralizing antibodies when introduced into mice in the form of a chimeric EtxB fusion protein. We suggest that fusion of peptide sequences to EtxB is a useful tool for the analysis of epitopes of complex proteins such as RTX toxins.

The importance in catalysis of the conserved arginine (R207) and lysine residues (K144, K294, K356, and K425) of 6-phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site-directed mutagenesis and kinetic analysis. In general mutations had minor effects on the Km for fructose 6-phosphate. More dramatic effects were seen on kcat. The R207A mutant had a five orders of magnitude decrease in kcat relative to the wild-type enzyme. There was a significant recovery, by three orders of magnitude, in the kcat for the R207K mutant. The results suggest that the positive charge provided by R207 plays a critical role in the isomerization reaction. K425 was substituted with alanine, valine, phenylalanine, tryptophan and aspartate. All mutant enzymes at position 425 had kcat decreased in the range of several-hundred-fold. For the other mutants, K294A and K144A, the kcat values were 3.5% and 27% of the wild-type enzyme, respectively. No effects on catalysis were observed for the K356A mutant. The results suggest that R207, K144, K294, and K425 are located in the active site of the enzyme. The active-site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5'-phosphate on enzymatic activities. The data also confirm the importance of K425 and K144 anticipated by the affinity labeling studies of the corresponding residues by pyridoxal 5'-phosphate in pig muscle phosphoglucose isomerase.

The initiation of replication from oriVRSF1010, the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB′ (primase), and RepC (intiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al. (1984) Proc. Natl. Acad. Sci. USA 81, 654–658; Scholz et al. (1985) in: Plasmids in Bacteria, pp. 243–259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp. 95–124, Academic Press, London; Scherzinger et al. (1991) Nucl. Acids Res. 19, 1203–1211]. We showed in this work that a newly constructed origin consisting of an oriVRSF1010 and a DnaA protein binding site, the dnaA box, inserted near oriVRSF1010 (oriVRSF1010-dnaA box) could function without RepB′ primase, but required RepA and RepC. This oriVRSF1010-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature. These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB′ dependent priming signals in oriVRSF1010, and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.

The replication origin of the broad host-range plasmid RSF1010 contains 3.5 copies of a 20mer iteron sequence that bind specifically to the plasmid-encoded initiator, RepC. Here we demonstrated that even a single iteron was bent upon binding of RepC. Moreover, the bending angle seems to become larger along with the increment of the number of iterons. In a mutational analysis of the iteron sequence, we isolated seven kinds of base-substitution mutants of iterons, and estimated the replication activity of these mutants in vivo. We found that each of the subsections in the 20mer iteron sequence made a distinct contribution to the initiation of RSF1010 DNA replication. With the binding assay of RepC and mutated iterons in vitro, we found that the formation of a productive RepC-iteron complex was required for the initiation of plasmid DNA replication.

To analyze the functional contribution to the ssiA function of subsections of the ssiA-determinant sequence based on their dimensions, we constructed ssiA mutants carrying insertions and deletions. Results of the examination of the ssiA mutants told us that, in addition to the base sequence, the dimensions were crucial factors for the functional contribution of the subsections of ssiA.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

The broad host range plasmid RSF1010 requires for its replication in Escherichia coli three plasmid-encoded proteins and specific nucleotide sequences ssiA, ssiB , and iterons in the oriV RSF1010 . In Pseudomonas aeruginosa , a recombinant mini-RSF1010 plasmid lacking ssiB lost its replication ability, but a miniplasmid lacking ssiA or carrying a primosome assembly site in place of ssiA could replicate. Moreover, ssiA , as a sole ssi signal, in the orientation thai ssiB had originally taken was sufficient for replication of the miniplasmid. These results indicated that only one RSF1010-specific ssi signal in the orientation that ssiB takes in wild-type oriV RSF1010 was essential for replication of RSF1010. Replication of the miniplasmids was dependent on the three plasmid-encoded proteins, RepA. B′, and C, as in E. coli.

Subunits of the heat-labile enterotoxin of Escherichia coli (LT) assemble in the periplasm and are secreted through the outer membrane in Vibrio cholerae. Deletions or substitutions of residues at the carboxyl terminus of the B subunit (EtxB) result in mutant polypeptides that assemble into normal pentamers at 30°C but cannot assemble at 42¼ in vivo. This defect may be suppressed by substitutions of single amino acid residues in regions that interact directly with the modified carboxyl terminus. Carboxyl terminal residues of EtxB thus appear to be required for formation or stabilization of an assembly intermediate of B subunit pentamerization but are not essential for the stability of the final pentamer.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.

Pleiotropic transposon insertion mutants of Vibrio cholerae that are unable to secrete enterotoxin, HA/protease and chitinase through the outer membrane have been isolated. The gene, epsM, responsible for complementation of two of the Tn5 insertion mutations was sequenced. It encodes a putative cytoplasmic membrane protein of 18.5 kDa that exhibits similarity to proteins required for extracellular secretion of pullulanase, pectate lyase or elastase in other Gram− bacteria. It is present on a 15-kb DNA fragment from the V. cholerae genome, containing the epsE gene that was previously shown to be required for secretion of cholera toxin [Sandkvist et al., Gene 123 (1993) 81–86]. Partial reading frames flanking epsM also demonstrated similarity to genes required for extracellular secretion of pullulanase in Klebsiella oxytoca.

To analyze the RSF1010-specific priming mechanism, a library of randomly mutagenized ssiA sequences was constructed by chemical synthesis using mixed nucleotide phosphoramidites. Synthetic ssiA sequences with the single base-substitutions were assayed for the SSI activity in E. coli JM109 expressing RepB' primase. It was demonstrated that the activity of ssiA was damaged markedly by single base-substitutions within the possible stem-loop structure and its 3'-flanking region. It is conceivable that these domains are critical in recognition and primer synthesis by RepB' primase.

A gene essential for the secretion of cholera toxin from the periplasm of Vibrio cholerae into the extracellular medium has been isolated and its nucleotide sequence determined. It encodes a cytoplasmic protein of 56 kDa that exhibits a high degree of similarity to gene products required for extracellular protein secretion in several other Gram− organisms. Sequence similarities in its potential ATP-binding site suggest that the protein may act as an energy provider or signal transducer in the process of extracellular secretion.

Xylans are linear polymers possessing a β-1,4-linked D-xylose backbone with branches containing 4-O-methyl D-glucuronic acid and l-arabinose. In several types of wood, particularly hardwoods, some of the sugar residues are acetylated (see Figure 33.1 for the schematic representation of the structure). Xylans or hemicelluloses are the second most abundant type of polysaccharides, after cellulose, in nature. As components of plant cell walls they constitute as much as 40% of all plant biomass (Timell, 1967; Aspinall, 1980; Gong et al., 1981). It is not surprising, therefore, that they are an attractive source of carbon and energy for microbes living in natural soil and aquatic habitats and that microorganisms have developed efficient ways to degrade these polymers and use their components.