Odile Possot

Multiple Interactions between Pullulanase Secreton Components Involved in Stabilization and Cytoplasmic Membrane Association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

Molecular characterization of PulE, a protein required for pullulanase secretion.

pulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide‐binding protein. Subcellular fractioNatlon indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non‐hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucteotide‐binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate‐rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide‐binding proteins. Altogether, these results Indicate that PulE might belong to a new family of nucleotide‐binding proteins. The protein could not be cross‐linked to the photoactivatable ATP analogue azido‐ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild‐type PulE protein. Evidence presented suggests that PulE might be a homodimer.

RECENT PROGRESS AND FUTURE DIRECTIONS IN STUDIES OF THE MAIN TERMINAL BRANCH OF THE GENERAL SECRETORY PATHWAY IN GRAM-NEGATIVE BACTERIA : A REVIEW

The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram- bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene

SummaryAn integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.

Pullulanase secretion in Escherichia coli K‐12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide‐binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytopLasmic protein. A representative PulE‐β‐galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF‐lacZ gene fusions that encoded hybrid proteins with relatively high levels of β‐galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K‐12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide‐binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway.

The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli. In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N‐terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to ≤10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m‐chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton‐motive force and to reduce lactose uptake to a significant extent.

The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin‐like PulG protein into a multiprotein complex

It has been proposed that the four type IV pilin‐like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans‐periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin‐like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X‐100 + EDTA or at high pH, but not by Triton X‐100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non‐sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross‐linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N‐methylates the PulG precursor, and PulE, a putative ATP‐binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.

Nucleotide sequence of regions homologous to nifH (nitrogenase Fe protein) from the nitrogen-fixing archaebacteria Methanococcus thermolithotrophicus and Methanobacterium ivanovii: evolutionary implications

SummaryDNA fragments bearing sequence similarity to eubacterialnifH probes were cloned from two nitrogen-fixing archaebacteria, a thermophilic methanogen,Methanococcus (Mc.) thermolithotrophicus, and a mesophilic methanogen,Methanobacterium (Mb.) ivanovii. Regions carrying similarities with the probes were sequenced. They contained several open reading frames (ORF), separated by A+T-rich regions. The largest ORFs in both regions, an 876-bp sequence inMc. thermolithotrophicus and a 789-bp sequence inMb.ivanovii, were assumed to be ORFsnifH. They code for polypeptides of mol. wt. 32,025 and 28,347, respectively. Both ORFsnifH were preceded by potential ribosome binding sites and followed by potential hairpin structures and by oligo-T sequences, which may act as transcription termination signals. The codon usage was similar in both ORFsnifH and was analogous to that used in theClostridium pasteurianum nifH gene, with a preference for codons ending with A or U. The ORFnifH deduced polypeptides contained 30% sequence matches with all eubacterialnifH products already sequenced. Four cysteine residues were found at the same position in all sequences, and regions surrounding the cysteine residues are highly conserved. Comparison of all pairs of methanogenic and eubacterialnifH sequences is in agreeement with a distant phylogenetic position of archaebacteria and with a very ancient origin ofnif genes. However, sequence similarity between Methanobacteriales and Methanococcales is low (around 50%) as compared to that found among eubacteria, suggesting a profound divergence between the two orders of methanogens. From comparison of amino acid sequences,C. pasteurianum groups with the other eubacteria, whereas comparison of nucleotide sequences seems to bringC. pasteurianum closer to methanogens. The latter result may be due to the high A+T content of bothC. pasteurianum and methanogens ORFsnifH or may come from an ancient lateral transfer betweenClostridium and methanogens.

PuIO, a component of the pullulanase secretion pathway of Klebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli

The PulO protein required for extracellular secretion of pullulanase by Klebsiella oxytoca is known to be highly homologous to two type IV prepilin peptidases, namely XcpA(PilD) (Pseudomonas aeruginosa) and TcpJ (Vibrio cholerae). The predicted prepilin peptidase activity of PulO was confirmed by showing that it could correctly process the product of the cloned pilE.1 type IV pilin structural gene from Neisseria gonorrhoeae in Escherichia coli. The P. aeruginosa prepilin peptidase and another putative prepilin peptidase, ComC from Bacillus subtilis, also processed prePilE. Subcellular fractionation showed that the pilE gene product that had been processed by PulO remained associated with the cytoplasmic membrane, as did the unprocessed precursor. PulO was also shown to process three of the four prePilE–PhoA hybrids tested. Southern hybridization experiments suggest that a PulO homologue is present in the N. gonorrhoeae chromosome.

THE CONSERVED TETRACYSTEINE MOTIF IN THE GENERAL SECRETORY PATHWAY COMPONENT PULE IS REQUIRED FOR EFFICIENT PULLULANASE SECRETION

The PulE component of the pullulanase secretion pathway, a typical main terminal branch of the general secretory pathway, has a tetracysteine motif (4Cys) that is also present in almost all of the many PulE homologues, including those involved in type-IV piliation and conjugal DNA transfer. The 4Cys resembles a zinc-binding motif found in other proteins such as adenylate kinases, which may be pertinent in view of the fact that PulE has a consensus ATP-binding motif and since at least one PulE homologue has been reported to have kinase activity. In PulE, the Cys residues of this motif form scrambled intra- and intermolecular disulfide bonds when cells are disrupted. Replacement of one or more Cys of this motif by Ser reduces PulE function, but at least two adjacent Cys must be replaced to prevent intramolecular disulfide bond formation.