Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein “Wzx-Wzy” system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.

Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located ␣-helices flanked by ␤-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. IMPORTANCEZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division. Bacterial cell division is an essential and complex process that requires the coordinated assembly of a multiprotein molecular machine termed the divisome. The divisome is responsible for constriction of the inner and outer membranes, synthesis of septal peptidoglycan, and subsequent septum formation. In Escherichia coli, divisome proteins are recruited in a hierarchical manner and can be divided into three main groups based on their order of assembly: (i) the proto-ring, (ii) early divisome proteins, and (iii) late divisome proteins (1, 2). The successful assembly of the divisome depends on the initial formation of the Z-ring, which is comprised of the 40-kDa bacterial tubulin homologue FtsZ. FtsZ assembles into filaments in a GTP-dependent manner and a headto-tail fashion (3-6). The filaments are then tethered to the membrane, forming the Z-ring. They act as the...

Using a special selection procedure, several mutants of Mycobacterium vaccae were isolated which were capable of converting sterols to 9 alpha-hydroxyandrost-4-ene-3,17-dion (9-OH-AD). Two mutants, Mycobacterium vaccae ZIMET 11052 and 11053, respectively, were further investigated. Strains of the species Mycobacterium fortuitum are mainly used for commercially obtaining 9-OH-AD from sterols. In contrast to the species Mycobacterium fortuitum the species Mycobacterium vaccae has not been reported to contain pathogenic strains. This seems an advantage for industrial application. Mutants with the ability of converting sterols to 9 alpha-hydroxysteroids have a defect in the steroid-1-dehydrogenase activity which is, however, only a partial one. The remaining activity may cause an undesirable degradation of the steroid nucleus. The steroid-1-dehydrogenase activity was tested using an assay developed by ATRAT (1986). We confirmed two apparently distinct steroid-1-dehydrogenases in Mycobacterium fortuitum NRRL B-8119 as reported by WOVCHA et al. (1979). One of them has an activity on androst-4-ene-3,17-dion (AD). The activity is increased by induction with sitosterol. The other one is active on 9-OH-AD. But Mycobacterium vaccae does not possess steroid-1-dehydrogenase activity on 9-OH-AD, and the AD specific steroid-1-dehydrogenase is not effected by sitosterol. The consequence is a high level of protection against steroid nucleus degradation yielding an effective accumulation of 9-OH-AD in fermentations with Mycobacterium vaccae mutants.

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In Escherichia coli, the N-terminal domain of the essential protein FtsK (FtsKN) is proposed to modulate septum formation through the formation of dynamic and essential protein interactions with both the Z-ring and late-stage division machinery. Using genomic mutagenesis, complementation analysis, and in vitro pull-down assays, we aimed to identify protein interaction partners of FtsK essential to its function during division. Here, we identified the cytoplasmic Z-ring membrane anchoring protein FtsA as a direct protein–protein interaction partner of FtsK. Random genomic mutagenesis of an ftsK temperature-sensitive strain of E. coli revealed an FtsA point mutation (G50E) that is able to fully restore normal cell growth and morphology, and further targeted site-directed mutagenesis of FtsA revealed several other point mutations capable of fully suppressing the essential requirement for functional FtsK. Together, this provides insight into a potential novel co-complex formed between these components during division and suggests FtsA may directly impact FtsK function.

Mutants with various levels of resistance to streptothricin were isolated from Escherichia coli K12, strain A19 after mutagenesis with N-methyl-N-nitro-N-nitroso-guanidine and ethylmethane-sulfonate. Nourseothricin, a mixture of streptothricin F and D was the selection agent. Spontaneous resistant mutants could not be found. The streptothricin-resistant mutant E. coli A19 Stcr 2/2/1 shows cross-resistance to some of the aminoglycoside antibiotics investigated, but no cross-resistance to chloramphenicol and chlortetracyclin. These results indicate similar mechanisms of action of streptothricin and aminoglycoside antibiotics.

Isolierung von Streptothricin-resistentenMutanten aus Escherichia coli K12, Stamm A19 L. 8EIDEL und INA HAUPT (Eingegangen urn 26. 4.1981) Mutants with various levels of resistance t o streptothricin were isolated from Escherichia coli K12, strain A19 after mutagenesis with N-methyl-N-nitro-N-nitroso-guanidine and ethylmethane-sulfonate. Nourseothricin, a mixture of streptothricin F and D was the selection agent. Spontaneous resistant mutants could not be found. The streptothricin-resistant mutant E. coli A19 Stcr 2/2/1 shows cross-resistance to some of the aminoglycoside antibiotics investigated, but no cross-resistance t o chloramphenicol and chlortetracyclin. These results indicate similar mechanisms of action of streptothricin and aminoglycoside antibiotics.