The Journal of General and Applied Microbiology Vol. 62(2016) No. 4

As similarly observed in nutrient-poor media at 37°C, Escherichia coli forms small rods in nutrient-rich media at temperatures near 8°C, the minimum temperature of growth. A study was initiated to identify proteins required to facilitate the small rod morphology at low temperature. E. coli contains three nonessential SPOR domain proteins (DamX, RlpA, and DedD) that have been demonstrated to bind to the septal ring. In contrast to the normal growth and small rod morphology of damX and rlpA null mutants at 10°C, the dedD null mutant exhibited reduced growth and formed filamentous cells. The presence of plasmid-encoded DedD restored growth and small rods. Plasmid-encoded FtsN, an essential SPOR domain protein that functions to stabilize the septal ring and to initiate septation, in the dedD null mutant resulted in increased growth and the formation of shorter chained cells. However, plasmid-encoded DedD failed to restore growth and cell division of cells lacking FtsN at 10°C. In contrast to cell division protein DedD, RodZ is a cell elongation protein particularly required for growth at 30°C. However, the rodZ null mutant grew similarly as the wild type strain and produced cocci in LB broth at 10°C. Moreover at 10°C, the concerted deletion of dedD and rodZ resulted in severe inhibition of growth accompanied with the formation of swollen prolate ellipsoids due to a block in septal ring assembly and cell elongation. The data indicate the cellular requirement of both FtsN and DedD for septation as well as RodZ for cell elongation to maintain the small rod morphology at temperatures near 8°C. In comparison to the growth and small rods of the wild type in M9-glucose minimal media at 37°C, the dedD null mutant grew at the same rate and produced elongated cells while the rodZ null mutant grew at a slightly slower rate and produced cocci. The data indicate that DedD and RodZ are also required to maintain the small rod morphology in nutrient-poor media, but there is a higher cellular requirement of DedD for growth and cell division in nutrient-rich media at low temperature.

It is the major characteristic of the cell-wall peptidoglycan structure in members of the family Micromonosporaceae that N-acetylmuramic acid (MurNAc) of glycan strand is replaced with N-glycolylmuramic acid (MurNGlyc). Consequently, it is difficult to use enzymatic methods for their peptidoglycan analyses. We therefore developed analysis method of peptidoglycan without using cell wall lytic enzymes as example to take the 3 genera, Micromonospora, Catenuloplanes, and Couchioplanes belonging to the family Micromonosporaceae, and their peptidoglycans were partially hydrolyzed with 4 M HCl at 60°C for 16 h followed by derivatization with N^α-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide (FDLA) or 1-phenyl-3-methyl-5-pyrazolone (PMP) and LC/MS analysis. Peptidoglycan of the genus Micromonospora consisted of a MurNGlyc–Gly–D-Glu–meso-diaminopimelyl (DAP)–D-Ala peptide stem and direct linkage between D-Ala and meso-DAP. In contrast, peptidoglycans of the genera Catenuloplanes and Couchioplanes consisted of a MurNGlyc–Gly–D-Glu–L-Lys–D-Ala peptide stem, and cross-linkage between D-Ala and L-Lys was mediated by an L-Ser residue. This method can be used to analyze the cell-wall peptidoglycan structure of other bacteria as well. By derivatization with FDLA or PMP followed by LC/MS analysis, the structure can be determined using only 0.2 mg of purified peptidoglycan.

Abandoned mine sites are frequently polluted with high concentrations of heavy metals. In this study, 25 calcite-forming bacteria were newly isolated from the soil of an abandoned metal mine in Korea. Based on their urease activity, calcite production, and resistance to copper toxicity, four isolates were selected and further identified by 16S rRNA gene sequencing. Among the isolates, Sporosarcina soli B-22 was selected for subsequent copper biosequestration studies, using the sand impermeability test by production of calcite and extracellular polymeric substance. High removal rates (61.8%) of copper were obtained when the sand samples were analyzed using an inductively coupled plasma-optical emission spectrometer following 72 h of incubation. Scanning electron microscopy showed that the copper carbonate precipitates had a diameter of approximately 5–10 μm. X-ray diffraction further confirmed the presence of copper carbonate and calcium carbonate crystals.