[show abstract][hide abstract] ABSTRACT: Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.
[show abstract][hide abstract] ABSTRACT: The lacZ gene from Escherichia coli has been used exten- sively as a reporter gene for studies of gene expression in Bacillus subtilis. These studies have sometimes been hindered by an endogenous b-galactosidase activity that is induced dur- ing sporulation (6, 8) and, in mutagenesis experiments, by regulatory mutations in a gene apparently encoding a repressor of the endogenous activity. We previously reported the isola- tion both of mutations in the regulatory gene, designated lacR, giving rise to increased levels of endogenous b-galactosidase and of secondary mutations in a linked gene which abolished the activity (9). The latter mutations, designated lacA, were thought to lie in the structural gene for the enzyme. The lacRA locus lies between hisA and thr, at about 290° (see reference 2) on the B. subtilis chromosome. A second, unlinked gene en- coding a cryptic endogenous b-galactosidase activity was re- ported by Zagorec and Steinmetz (20). Here we report the isolation of transposon insertions in the lacA and lacR genes, the DNA sequence of the region, and the construction and characterization of strains with defined insertions in the two genes. Transposon mutagenesis was done with the mini-Tn10 cat plasmid pHV1248 (see Table 1 for bacterial strains and plas- mids). The plasmid was introduced into B. subtilis SG68, which carries a mutation in the lacR gene leading to constitutive levels of endogenous b-galactosidase and hence blue colonies on indicator plates containing X-Gal (5-bromo-4-chloro-3-in- dolyl-b-D-galactopyranoside) (9). The strain was incubated at the nonpermissive temperature for plasmid replication on plates maintaining selection for the mini-Tn10-borne chloram- phenicol resistance determinant. Three white colonies were obtained among approximately 5,000 colonies. Transformation of DNA from each of the chloramphenicol- resistant strains back into SG68 showed that the transposon insertion was completely linked to the mutation giving rise to the white colony phenotype. To recover DNA adjacent to the transposon insertion, one of the new strains, SG80, was trans- formed with plasmid pRD110. Selection for resistance to spec- tinomycin (50 mg/ml) led to insertion of the plasmid into the transposon by homologous recombination with the cat gene. Plasmids with inserts extending out from the transposon were
[show abstract][hide abstract] ABSTRACT: Systematic inactivation of Bacillus subtilis genes has previously revealed that 271 are indispensable for growth. In the present study, 11 of these (yacA, ydiB, ydiC, ykqC, ylaN, yloQ, ymdA, yneS, yqeI, yqjK and ywlC) were identified as genes encoding proteins of unknown function. By analysing the effects of protein depletion, and examining the subcellular localization of these proteins, a start has been made in elucidating their functions. It was found that four of these genes (ydiB, yloQ, yqeI and ywlC) were not required for B. subtilis viability. Analysis of the localization of YkqC suggests that it co-localizes with ribosomes, and it is proposed that it is involved in processing either rRNA or specific mRNAs when they are associated with the ribosome. The results suggest that other novel essential proteins may be involved in lipid synthesis and control of cell wall synthesis.