Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth.     

Identification of the Escherichia coli cell division gene sep and organization of the cell division-cell envelope genes in the sep-mur-ftsA-envA cluster as determined with specialized transducing lambda bacteriophages.

From a lysogen with lambda integrated in the leu operon, specialized transducing phages that carry the cell division, murein biosynthesis, and envelope permeability genes located about 0.5 min to the right of leu were isolated. These phages were used to identify the previously undiscovered cell division gene sep. A genetic map proves that sep is located in the sequence leuA sep murE murF murC ddl ftsA envA. A physical map of this region was prepared by heteroduplex analysis of the phage DNAs. Overlapping segments of host DNA extended rightward for as much as 26.4 kilobase pairs from the prophage insertion point (thought to be in leuA) to include all the genes through envA.     

The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes

We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes.     

Two new mutant loci (smhB and lytD) in Escherichia coli which confer temperature-sensitive growth and lysis phenotypes

A temperature-sensitive mutation in the murH gene of Escherichia coli confers a lysis phenotype at the restrictive temperature. An extragenic suppressor of murH apparently representing a new locus at 12.5 min on the linkage map and designated smhB is described. The smhB mutation by itself also conferred a temperature-sensitive lysis phenotype. A mutation in another new locus designated lytD which arose spontaneously in the smhB mutant was mapped close to smhB at 12.7 min on the linkage map. The lytD mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. Thus, the suppression of lysis in the smhB murH and the smhB lytD double mutants suggests a mechanism involving the reciprocal suppression of the two individual lysis-causing mutant alleles. The suppressor activity of smhB was apparently relatively specific in that smhB failed to prevent lysis induced by either mutational (murE or murF) or antibiotic-induced blocks in peptidoglycan synthesis. This suggests that murH, smhB, and lytD may be functionally related.     

Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.     

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

A method for demonstrating gene essentiality in Staphylococcus aureus

A method for demonstrating whether a gene of Staphylococcus aureus is essential for growth in a rich medium is described. We have used this method to determine whether the murE gene, which encodes the UDP-N-acetylmuramyl tripeptide synthetase required for peptidoglycan synthesis, is essential for growth in S. aureus. In this study, strain CYL368 was constructed from S. aureus RN4220 by placing the murE gene in the chromosome under the control of the spac promoter (a hybrid promoter of the Escherichia coli lac operator and the Bacillus subtilis SPO1 phage promoter). To regulate the murE gene in CYL368, the E. coli lacI gene was expressed from the B. licheniformis penicillinase gene (pcn) promoter in plasmid pMJ8426. Strain CYL368(pMJ8426) grew normally in the presence of isopropyl-beta-d-thiogalactopyranoside but could not grow in the absence of the inducer. These results indicate that the murE gene expressed from the spac promoter in CYL368(pMJ8426) is needed for bacterial growth. We concluded that murE is an essential gene of S. aureus.     

New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.     

Point mutations in a peptidoglycan biosynthesis gene cause competence induction in Haemophilus influenzae

We have identified three new Haemophilus influenzae mutations causing cells to exhibit extreme hypercompetence at all stages of growth. The mutations are in murE, which encodes the meso-diaminopimelate-adding enzyme of peptidoglycan synthesis. All are point mutations causing nonconservative amino acid substitutions, two at a poorly conserved residue (G(435)-->R and G(435)-->W) and the third at a highly conserved leucine (L(361)-->S). The mutant strains have very similar phenotypes and do not exhibit any defects in cell growth, permeability, or sensitivity to peptidoglycan antibiotics. Cells retain the normal specificity of DNA uptake for the H. influenzae uptake signal sequence. The mutations do not bypass genes known to be needed for competence induction but do dramatically increase expression of genes required for the normal pathway of DNA uptake. We conclude that the mutations do not act by increasing cell permeability but by causing induction of the normal competence pathway via a previously unsuspected signal.     

Nucleotide sequence of the murE gene of Escherichia coli

The nucleotide sequence of the murE gene encoding the diaminopimelic acid adding enzyme of Escherichia coli is reported. The coding region consisted of 1413 base pairs and was separated from the ftsI (penicillin-binding protein 3) gene by 61 base pairs. The deduced primary structure of MurE comprised 471 amino acid residues with a molecular mass of 50.6 kilodaltons.     

Streptococcus gordonii utilizes several distinct gene functions to recruit Porphyromonas gingivalis into a mixed community

Dental plaque biofilm formation proceeds through a developmental pathway initiated by the attachment of pioneer organisms, such as Streptococcus gordonii, to tooth surfaces. Through a variety of synergistic interactions, pioneer organisms facilitate the colonization of later arrivals including Porphyromonas gingivalis, a potential periodontal pathogen. We have investigated genes of S. gordonii required to support a heterotypic biofilm community with P. gingivalis. By screening a plasmid integration library of S. gordonii, genes were identified that are crucial for the accumulation of planktonic P. gingivalis cells into a multispecies biofilm. These genes were further investigated by specific mutation and complementation analyses. The biofilm-associated genes can be grouped into broad categories based on putative function as follows: (i) intercellular or intracellular signalling (cbe and spxB), (ii) cell wall integrity and maintenance of adhesive proteins (murE, msrA and atf), (iii) extracellular capsule biosynthesis (pgsA and atf), and (iv) physiology (gdhA, ccmA and ntpB). In addition, a gene for a hypothetical protein was identified. Biofilm visualization and quantification by confocal microscopy confirmed the role of these genes in the maturation of the multispecies community, including biofilm architectural development. The results suggest that S. gordonii governs the development of heterotypic oral biofilms through multiple genetic pathways.     

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.     

Identification of the amidotransferase AsnB1 as being responsible for meso-diaminopimelic acid amidation in Lactobacillus plantarum peptidoglycan

The peptidoglycan (PG) of Lactobacillus plantarum contains amidated meso-diaminopimelic acid (mDAP). The functional role of this PG modification has never been characterized in any bacterial species, except for its impact on PG recognition by receptors of the innate immune system. In silico analysis of loci carrying PG biosynthesis genes in the L. plantarum genome revealed the colocalization of the murE gene, which encodes the ligase catalyzing the addition of mDAP to UDP-N-muramoyl-d-glutamate PG precursors, with asnB1, which encodes a putative asparagine synthase with an N-terminal amidotransferase domain. By gene disruption and complementation experiments, we showed that asnB1 is the amidotransferase involved in mDAP amidation. PG structural analysis revealed that mDAP amidation plays a key role in the control of the l,d-carboxypeptidase DacB activity. In addition, a mutant strain with a defect in mDAP amidation is strongly affected in growth and cell morphology, with filamentation and cell chaining, while a DacB-negative strain displays a phenotype very similar to that of a wild-type strain. These results suggest that mDAP amidation may play a critical role in the control of the septation process.     

Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D- Ala, has been purified to homogeneity from an E. coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF. The enzyme is a monomer of molecular weight 49,000, and it has a turnover number of 784 min-1 for ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelyl-D-++ +trifluoroAla-D- trifluoroAla.