Cloning and Characterization of Polyhydroxybutyrate Synthase from Methylobacterium extorquens AM1

В результате поиска генов, кодирующих вероятные ПГБ-синтазы в геномах бактерий рода Methylobacterium, выявлены множественные (до пяти у одного штамма) гены ПГБ-синтаз. Филогенетическим анализом показано, что белки PhaC1, PhaC2, PhaC3 относятся к I классу ПГБ-синтаз, белки PhaC4 – к ПГБ-синтазам III класса, тогда как PhaC5, по-видимому, представляет неохарактеризованный класс ПГБ-синтаз. Впервые выделена и охарактеризована рекомбинантная ПГБ-синтаза I класса (КФ 2.3.1.B2) из Methylobacterium extorquens AM1, кодируемая геном phaC1. Молекулярная масса мономера фермента составила 78 кДа. Константа Михаэлиса (Km) для PhaC1 из штамма AM1 составила 1,3 мМ, а максимальная скорость реакции (Vmax) – 0,1 мкмоль∙мин-1∙мг-1. Получен делеционный мутант Methylobacterium extorquens по гену phaC, перспективный для дальнейшего исследования особенностей биосинтеза ПГБ метилобактериямиMultiple genes encoding putative PHB synthases (up to 5 in single strain) were found in Methylobacterium genomes. As a result of phylogenetic analysis proteins PhaC1, PhaC2, PhaC3 were identified as class I PHB synthases, PhaC4 proteins were identified as class III PHB synthases, while PhaC5 apparently belongs to uncharacterized class of PHB synthases. Firstly, the recombinant class I PBH synthase (EC 2.3.1.B2) encoded by phaC1 gene from Methylobacterium extorquens AM1 was purified and characterized. Molecular mass of enzyme monomer was 78 kDa. Michaelis constant (Km) for PhaC1 was 1,3 mM and maximal reaction rate (Vmax) was 0,1 μmol∙min–1∙mg–1. The deletion mutant of Methylobacterium extorquens in the phaC gene was obtained which is promising for further study of peculiarities of methylobacteria’s PHB biosynthesi

Modification of cell wall polysaccharide guides cell division in <i>Streptococcus mutans</i>

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning. [Figure not available: see fulltext.

PplD Is a De-N-Acetylase of the Cell Wall Linkage Unit of Streptococcal Rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins

Cloning and Characterization of Polyhydroxybutyrate Synthase from Methylobacterium extorquens AM1

В результате поиска генов, кодирующих вероятные ПГБ-синтазы в геномах бактерий рода Methylobacterium, выявлены множественные (до пяти у одного штамма) гены ПГБ-синтаз. Филогенетическим анализом показано, что белки PhaC1, PhaC2, PhaC3 относятся к I классу ПГБ-синтаз, белки PhaC4 – к ПГБ-синтазам III класса, тогда как PhaC5, по-видимому, представляет неохарактеризованный класс ПГБ-синтаз. Впервые выделена и охарактеризована рекомбинантная ПГБ-синтаза I класса (КФ 2.3.1.B2) из Methylobacterium extorquens AM1, кодируемая геном phaC1. Молекулярная масса мономера фермента составила 78 кДа. Константа Михаэлиса (Km) для PhaC1 из штамма AM1 составила 1,3 мМ, а максимальная скорость реакции (Vmax) – 0,1 мкмоль∙мин-1∙мг-1. Получен делеционный мутант Methylobacterium extorquens по гену phaC, перспективный для дальнейшего исследования особенностей биосинтеза ПГБ метилобактериямиMultiple genes encoding putative PHB synthases (up to 5 in single strain) were found in Methylobacterium genomes. As a result of phylogenetic analysis proteins PhaC1, PhaC2, PhaC3 were identified as class I PHB synthases, PhaC4 proteins were identified as class III PHB synthases, while PhaC5 apparently belongs to uncharacterized class of PHB synthases. Firstly, the recombinant class I PBH synthase (EC 2.3.1.B2) encoded by phaC1 gene from Methylobacterium extorquens AM1 was purified and characterized. Molecular mass of enzyme monomer was 78 kDa. Michaelis constant (Km) for PhaC1 was 1,3 mM and maximal reaction rate (Vmax) was 0,1 μmol∙min–1∙mg–1. The deletion mutant of Methylobacterium extorquens in the phaC gene was obtained which is promising for further study of peculiarities of methylobacteria’s PHB biosynthesi