In present work, we elucidate the GlcNAc metabolic machinery in strain and demonstrated the role of nagA in GlcNAc metabolism by cloning a DNA fragment encoding a nagA and subsequently generating a nagA-deficient mutant by homologous recombination. The resulting knockout strain was examined for growth, cytoplasmic UDPGlcNAc pool, and overall cellulose productivity with glucose and/ or GlcNAc as a carbon source. The successful deletion of this gene and the subsequent analysis provides a clearer picture of the related metabolic pathways of this potentially important biosynthetic pathway. To determine whether nagA is essential for growth of the bacterium, both DnagA and wild type G. xylinus were grown for five days on HS agar plate supplemented with either glucose, GlcNAc, or glucosamine. On glucose and glucosamine supplemented plates, both mutant and wild type grew well while on GlcNAc supplemented plates, mutant cells did not grow. To confirm these data, the growth of both wild type and mutant cells was monitored in liquid HS media supplemented with glucose or GlcNAc. In glucose-supplemented media, both mutant and wild type cells exhibited a typical sigmoidal growth pattern whereas in the presence of GlcNAc, growth of mutant cells was completely inhibited while wild type cells grew slowly as expected due to the fact that GlcNAc is not a preferred carbon source for G. xylinus. Additionally, in presence of glucosamine, growth of both wild type and DnagA mutant was similar to the growth of wild type in GlcNAc fed conditions. These findings together with agar plate growth studies demonstrated that nagA disruption prevents G. xylinus from metabolizing GlcNAc as an alternative carbon source for growth. Similar findings were also observed in Gluconacetobacter intermedius where disruption of nagA decreased the growth rate in the exponential growth phase. Nevertheless, a steady sate growth curve of mutant cells with GlcNAc feed also revealed that nagA disruption does not cause any lethal impact on the bacterial cells and as a result it only impairs the growth. To conform the mutant cells were sub-cultured in the presence of glucose and found that bacterium regains its normal growth.
Month: September 2018
Before clearance of the inducer become more easily subjected to heterochro
For instance, in addition to the effect of temperature Cu concentrations of approximately 4.4 mg L21 and 0.4 mg L21 lead to a further 10% reduction in metamorphosis success for Acropora millepora larvae at 32uC and 33uC respectively, and Cu concentrations of approximately 14.8 mg L21 and 10.4 mg L21 caused the same effect for A. tenuis larvae at these temperatures. In fact, Acropora larvae tested in this study were as susceptible to Cu at 32uC deg as Mytilus edulis embryos, the most sensitive marine organisms previously reported. The monsoon season poses the greatest challenge to the survival of nearshore coral communities. During this period heavy rains transport sediments from land to sea bringing the highest levels of water contamination that are experienced over the annual cycle. Coral spawning typically occurs after the full moon in the month before maximum rainfall and can coincide with high SSTs. The average monthly temperature during coral spawning in Nelly Bay where the corals for this study were collected ranged from 26.3 to 29.8uC. However, synergistic interactions between temperature and Cu occurred in the present study at and above 31uC for both species indicating that greater than additive effects of these stressors are presently unlikely to be experienced at this site except for that proportion of larvae that show delayed metamorphosis. Peak rainfall in the GBR catchments is from December to March, but early rainfall of the monsoon season in November and December may also deliver the high loads of pollution into the GBR lagoon. Although SSTs thresholds for coral larvae and the highest loads of river-borne pollution do not usually coincide with the major coral spawning events, they are often only a month apart and this may become more of a concern as climate change impacts upon SST and rainfall patterns in the tropics. SSTs are projected to increase by between 1.8–4.0uC by 2100, potentially impacting upon the success of coral recruitment, especially if pollution thresholds are exceeded.