A CRISPR with roles in Myxococcus xanthus development and exopolysaccharide production.

The Gram-negative soil bacterium Myxococcus xanthus utilizes its social (S) gliding motility to move on surfaces during its vegetative and developmental cycles. It is known that S motility requires the type IV pilus (T4P) and the exopolysaccharide (EPS) to function. The T4P is the S motility motor, and it powers cell movement by retraction. As the key regulator of the S motor, EPS is proposed to be the anchor and trigger for T4P retraction. The production of EPS is regulated in turn by the T4P in M. xanthus, and T4P(-) mutants are S(-) and EPS(-). In this study, a ΔpilA strain (T4P(-) and EPS(-)) was mutagenized by a transposon and screened for EPS(+) mutants. A pilA suppressor isolated as such harbored an insertion in the 3rd clustered regularly interspaced short palindromic repeat (CRISPR3) in M. xanthus. Evidence indicates that this transposon insertion, designated CRISPR3*, is a gain-of-function (GOF) mutation. Moreover, CRISPR3* eliminated developmental aggregation in both the wild-type and the pilA mutant backgrounds. Upstream of CRISPR3 are genes encoding the repeat-associated mysterious proteins (RAMPs). These RAMP genes are indispensable for CRISPR3* to affect development and EPS in M. xanthus. Analysis by reverse transcription (RT)-PCR suggested that CRISPR3* led to an increase in the processing of the RNA transcribed from CRISPR3. We propose that certain CRISPR3 transcripts, once expressed and processed, target genes critical for M. xanthus fruiting body development and EPS production in a RAMP-dependent manner.

Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates.

The biocompatibility of microbial polyesters polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) were evaluated in vitro. The mouse fibroblast cell line L929 was inoculated on films made of PHB, PHBHHx and their blends, polylactic acid (PLA) as control. It was found that the growth of the cells L929 was poor on PHB and PLA films. The viable cell number ranged from 8.8 x 10(2) to 1.8 x 10(4)/cm2 only. Cell growth on the films made by blending PHB and PHBHHx showed a dramatic improvement. The viable cell number observed increased from 9.7 x 10(2) to 1.9 x 10(5) on a series of PHB/PHBHHx blended film in ratios of 0.9/0.1:0/1, respectively, indicating a much better biocompatibility in the blends contributed by PHBHHx. Biocompatibility was also strongly improved when these polymers were treated with lipases and NaOH, respectively. However, the effects of treatment were weakened when PHBHHx content increased in the blends. It was found that lipase treatment had more increased biocompatibility than NaOH. After the treatment biocompatibility of PHB was approximately the same as PLA, while PHBHHx and its dominant blends showed improved biocompatibility compared to PLA.