Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-()-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.     

Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-(γ)-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.     

Analysis of a Streptomyces coelicolor A3(2) locus containing the nucleoside diphosphate kinase (ndk) and folylpolyglutamate synthetase (folC) genes

A 3.6-kb DNA fragment from Streptomyces coelicolor A3(2) with the genes valS probably encoding a valyl-tRNA synthetase, folC encoding folylpolyglutamate synthetase, and ndk encoding a nucleoside diphosphate kinase was analysed. folC and ndk are separated by a small open reading frame of unknown function, orfX. The deduced folC gene product is a protein of 46 677 Da whose sequence is similar to other folylpolyglutamate synthetases and folylpolyglutamate synthetase-dihydrofolate synthetases from both Gram-positive and Gram-negative bacteria. After cloning folC behind the lacZ promoter, the Streptomyces folC complemented a folC mutant of Escherichia coli. An essential function for Streptomyces folC was suggested by the fact that it could not be mutated using a conventional gene disruption technique.     

Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli

On the basis of the published N-terminal amino acid sequence of the soluble lytic transglycosylase 35 (Slt35) of Escherichia coli, an open reading frame (ORF) was cloned from the 60.8 min region of the E. coli chromosome. The nucleotide sequence of the ORF, containing a putative lipoprotein-processing site, was shown by [³H]-palmitate labelling to encode a lipoprotein with an apparent molecular mass of 36 kDa. A larger protein, presumably the prolipoprotein form, accumulated in the presence of globomycin. Over-expression of the gene, designated mltB (for membrane-bound lytic transglycosylase B), caused a 55-fold increase in murein hydrolase activity in the membrane fraction and resulted in rapid cell lysis. After membrane fractionation by sucrose-density-gradient centrifugation, most of the induced enzyme activity was present in the outer and intermediate membrane fractions. Murein hydrolase activity in the soluble fraction of a homogenate of cells induced for MltB increased with time. This release of enzyme activity into the supernatant could be inhibited by the addition of the serine-protease inhibitor phenylmethyl-sulphonyl fluoride. It is concluded that the previously isolated Slt35 protein is a proteolytic degradation product of the murein hydrolase lipoprotein MltB. Surprisingly, a deletion in the mltB gene showed no obvious phenotype.     

Studies on the metabolic role of peptidyl-tRNA hydrolase

Summary A mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp ⁺ (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.Journal paper No. J-7465 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1747.     

The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.     

Cloning and characterization of a bile salt hydrolase (<Emphasis Type="Italic">bsh</Emphasis>) from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis>

A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P_bsh) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.     

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells

Summary Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk^- cells as well as E. coli tk^- cells to the Tk⁺ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk ^- mutant of E. coli C600.     

Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae – a review

In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.     

Thymol-triggered lysis of Escherichia coli expressing Lactobacillus phage LL-H muramidase

Effective disruption of Escherichia coli cells is achieved by the intracellularly accumulated recombinant murein hydrolase (Lactobacillus bacteriophage LL-H muramidase) after the addition of 5 mM thymol. Thymol destroys the integrity and electric potential of the cytoplasmic membrane, and as a consequence the muramidase can access and hydrolyze the cell wall murein leading to cell lysis. Lysis occurred within 5 min after the addition of thymol and seemed to be efficient at high culture densities. This lysis method does not require cell harvesting or addition of other cell wall weakening substances or exogenous enzymes. As a cell disruption method, thymol-triggered lysis is as efficient as sonication in the presence of 1% Triton. Furthermore, thymol did not interfere with the purification steps of Mur by expanded bed adsorption chromatography (EBA), suggesting that the lysis method presented here is well suited for large-scale production and purification of intracellular proteins of E. coli. Received 21 April 1998/ Accepted in revised form 5 December 1998     

Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis.

Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 of Listeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-tagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by EsigmaD RNA polymerase. Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

State of the rigid-layer in cell walls of some gram-negative bacteria

Summary Rigid-layers in cell walls of Enterobacteriaceae and Pseudomonadales contain murein sacculi of identical chemotype. However, lipoprotein in covalent linkage to the murein participates to a different extent in rigid layer construction in the two taxonomic groups. In Proteus mirabilis and Escherichia coli lipoprotein particles of similar size and spatial arrangement are visible in the electron microscope as major rigid layer components. In the cell walls of Pseudomonadales covalent (lipo-)protein plays a far less prominent role. It is present as a minor constituent in rigid layers of Pseudomonas aeruginosa. Rigid layers of Spirillum serpens are naked murein sacculi devoid of any covalently attached protein.     

The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine methyltransferase enzyme confers a specific DNA methylation pattern in an McrBC-deficient Escherichia coli strain

The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced M_r of 40 700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M·NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.     

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.     

Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli

We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.     

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

Summary A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27 513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M _r value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M _r value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.