Molecular Microbiology

Berks, Ben C.

doi: 10.1046/j.1365-2958.1996.00114.xpmid: 8939424

The precursor polypeptides of periplasmic proteins binding seven types of redox cofactor have unusually long signal sequences bearing a consensus (S/T)‐R‐R‐x‐F‐L‐K motif immediately before the hydrophobic region. Such ‘double‐arginine’ signal sequences are not, in general, found on the precursors of other periplasmic proteins. It is suggested that precursor proteins with double‐arginine signal sequences share a common specialization in their export pathway. The nature of this specialization, the structure of the double‐arginine signal sequences, and the possible relationship with the double‐arginine signal peptide‐dependent thylakoid import pathway are discussed.

Leonardo, Michael R.; Forst, Steven

doi: 10.1046/j.1365-2958.1996.1271487.xpmid: 8939425

In Escherichia coli, EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR. OmpR‐phosphate regulates the expression of the porin genes, ompF and ompC. To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted. Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity. The periplasmic domain of EnvZ was also replaced with the non‐homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis. Osmoregulation of OmpF and OmpC production in cells containing the PhoR–EnvZ hybrid protein was indistinguishable from that in cells containing wild‐type EnvZ. Identical results were obtained with an envZ–pta/ack strain, which could not synthesize acetyl phosphate. Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study. These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.

McGowan, S. J.; Sebaihia, M.; Porter, L. E.; Stewart, G. S. A. B.; Williams, P.; Bycroft, B. W.; Salmond, G. P. C.

doi: 10.1046/j.1365-2958.1996.00125.xpmid: 28777504

Carbapenems are β‐lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug‐resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora, makes the simple β‐lactam antibiotic, 1‐carbapen‐2‐em‐3‐carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the β‐lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so‐called sulphur‐containing β‐lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC, encode proteins which show significant homology with proteins encoded by the Streptomycesclavuligerus gene cluster responsible for the production of the β‐lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the β‐lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β‐lactam‐ring‐containing antibiotics.

Sourjik, Victor; Schmitt, Rüdiger

doi: 10.1046/j.1365-2958.1996.1291489.xpmid: 8939427

Cells of Rhizobium meliloti swim by the unidirectional, clockwise rotation of their right‐handed helical flagella and respond to tactic stimuli by modulating the flagellar rotary speed. We have shown that wild‐type cells respond to the addition of proline, a strong chemoattractant, by a sustained increase in free‐swimming speed (chemokinesis). We have examined the role of two response regulators, CheY1 and CheY2, and of CheA autokinase in the chemotaxis and chemokinesis of R. meliloti by comparing wild‐type and mutant strains that carry deletions in the corresponding genes. Swarm tests, capillary assays, and computerized motion analysis revealed that (i) CheY2 alone mediates 60 to 70% of wild‐type taxis, whereas CheY1 alone mediates no taxis, but is needed for the full tactic response; (ii) CheY2 is the main response regulator directing chemokinesis and smooth swimming in response to attractant, whereas CheY1 contributes little to chemokinesis, but interferes with smooth swimming; (iii) in a CheY2‐overproducing strain, flagellar rotary speed increases upon addition and decreases upon removal of attractant; (iv) both CheY2 and CheY1 require phosphorylation by CheA for activity. We conclude that addition of attractant causes inhibition of CheA kinase and removal causes activation, and that consequent production of CheY1‐P and CheY2‐P acts to slow the flagellar motor. The action of the chief regulator, CheY2‐P, on flagellar rotation is modulated by CheY1, probably by competition for phosphate from CheA.

Titheradge, Annette J. B.; Ternent, Diane; Murray, Noreen E.

doi: 10.1046/j.1365-2958.1996.00126.xpmid: 28777503

Salmonella enterica serovar blegdam has a restriction and modification system encoded by genes linked to serB. We have cloned these genes, putative alleles of the hsd locus of Escherichia coli K‐12, and confirmed by the sequence similarities of flanking DNA that the hsd genes of S. enterica serovar blegdam have the same chromosomal location as those of E. coli K‐12 and Salmonella enterica serovar typhimurium LT2. There is, however, no obvious similarity in their nucleotide sequences, and while the gene order in S. enterica serovar blegdam is serB hsdM, S and R, that in E. coli K‐12 and S. enterica serovar typhimurium LT2 is serB hsdR, M and S. The hsd genes of S. enterica serovar blegdam identify a third family of serB‐linked hsd genes (type ID). The polypeptide sequence predicted from the three hsd genes show some similarities (18–50% identity) with the polypeptides of known and putative type I restriction and modification systems; the highest levels of identity are with sequences of Haemophilus influenzae Rd. The HsdM polypeptide has the motifs characteristic of adenine methyltransferases. Comparisons of the HsdR sequence with those for three other families of type I systems and three putative HsdR polypeptides identify two highly conserved regions in addition to the seven proposed DEAD‐box motifs.

Chambers, Alistair

doi: 10.1046/j.1365-2958.1996.00134.xpmid: 8939429

Budding yeast (Saccharomyces cerevisiae) Rap1p has been expressed in fission yeast (Schizosaccharo‐myces pombe) under the control of the regulatable fructose bisphosphatase (fbp) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature‐sensitive mutation in the DNA‐binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1pts is severely inhibited. Removal of a short region downstream of the DNA‐binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.

Moeck, Gregory S.; Tawa, Paul; Xiang, Hui; Ismail, Ashraf A.; Turnbull, Joanne L.; Coulton, James W.

doi: 10.1046/j.1365-2958.1996.00112.xpmid: 8939430

Ferrichrome–iron is actively transported across the outer membrane of Escherichia coli by the TonB‐dependent receptor FhuA. To obtain FhuA in a form suitable for secondary‐structure analyses, a hexahistidine tag was inserted into a surface‐located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand‐binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of ‐sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti‐FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N‐terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.

Summers, Michael L.; Meeks, John C.

doi: 10.1046/j.1365-2958.1996.1371502.xpmid: 8939431

The gene encoding glucose‐6‐phosphate dehydrogenase (G6PD), zwf, in Nostocpunctiforme strain ATCC 29133 is part of a four‐gene operon that also encodes fructose bisphosphatase (fbp), transaldolase (tal ) and a gene product termed OpcA, which is cotranscribed with zwf and essential for G6PD activity. The effect of exogenous nitrogen and carbon sources on transcription of these genes was investigated. Growth in the presence of ammonium yielded low levels of transcripts encoding all genes of the operon, while growth under nitrogen‐fixing conditions resulted in a large increase of transcripts encoding for fbp and zwf–opcA. When cells are grown in the presence of fructose, levels of transcripts encoding tal and zwf–opcA were increased, relative to levels in ammonium‐grown cells. These results indicate that this facultatively heterotrophic cyanobacterium can respond to changes in its environment by altering transcription of genes involved in carbon catabolism. Primer extension identified five 5′ ends corresponding to the major regulated transcripts which we conclude arise from independent transcriptional start points.

Burrows, L. L.; Charter, D. F.; Lam, J. S.

doi: 10.1046/j.1365-2958.1996.1351503.xpmid: 8939432

Pseudomonas aeruginosa co‐expresses A‐band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B‐band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype‐specific antigen. The gene clusters for A‐ and B‐band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B‐band O‐antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA–wbpN. A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O‐antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup‐specific, knockout mutations confirmed it is necessary for O5 O‐antigen biosynthesis. A novel insertion sequence, IS1209, is present at the junction between the serogroup‐specific and non‐specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O‐antigen biosynthesis.

Song, Jian; Jensen, Roy A.

doi: 10.1046/j.1365-2958.1996.00131.xpmid: 8939433

Pseudomonas aeruginosa was recently found to possess a cluster of structural genes encoding phenylalanine hydroxylase (PhhA), carbinolamine dehydratase (PhhB), and aromatic aminotransferase (PhhC). We now report the presence, in the flanking upstream region, of a divergently transcribed gene (phhR ) encoding an activator protein. Inactivation of phhR markedly reduced expression of the structural genes. PhhR belongs to the large prokaryote family of 54 enhancer‐binding proteins, and activation of the phh operon by PhhR in P. aeruginosa required rpoN. The closest homologues of PhhR are the TyrR proteins from Escherichia coli and Haemophilus influenzae. E. coli TyrR is an unusual member of the homologue family in that the transcriptional units regulated by tyrR are driven by 70 promoters. P. aeruginosaphhR was able to replace E. coli tyrR as a repressor of the aroF–tyrA operon (but not as an activator of mtr ) in the heterologous E. coli system. Two regions that resemble E. coli TyrR boxes were identified in the intervening region between phhR and phhA. We propose that one or both boxes may be the target of PhhR acting as an autogenous repressor at a 70 promoter in one direction. In the other direction, one or both boxes may be the upstream activator sequence targeted by PhhR to facilitate expression of the phh operon from a 54 promoter. The phh operon was strongly induced in fructose‐ or glucose‐based minimal medium by l‐phenylalanine. Inactivation of phhR in P. aeruginosa abolished ability to utilize either l‐phenylalanine or l‐tyrosine as a sole source of carbon for growth.