Experimental Study on the Compaction Characteristics and Evaluation Method of Coarse-Grained Materials for Subgrade.

Coarse-grained materials are widely used in high-speed railway construction, and it is of great significance to research its compaction characteristics due to the high quality control requirements. In this regard, a field compaction experiment was conducted at a subgrade near Bazhou Station of Beijing-Xiong'an Intercity Railway. The test results of the compaction effect were presented in this study at first. The roller-integrated compaction measurements (i.e., compaction meter value, CMV) were compared with several traditional in-situ tests (i.e., plate load test, light falling weight deflectometer test, and shear wave velocity test). Then the stability of CMV was evaluated by the proposed δ criterion. The spatial uniformity of compaction was further investigated. Based on the analysis, the target value of CMV was preliminarily determined. It showed that Evd was more variable than CMV. The results convincingly indicated that the compaction parameters increased with the increasing number of roller passes at first. A further increase in compaction effort could result in the decompaction of material when the compaction number up to a certain value. The stability analysis method proposed in this study showed its potency of quantifying the percentage of areas with acceptable compaction. The geostatistical analysis could reflect the spatial uniformity of compaction. Overall, the conducted study could provide a useful reference for geo-material compaction control in the transportation engineering.

Transcriptome and Zymogram Analyses Reveal a Cellobiose-Dose Related Reciprocal Regulatory Effect on Cellulase Synthesis in Cellulosilyticum ruminicola H1.

The rumen bacterium Cellulosilyticum ruminicola H1 efficiently hydrolyzes cellulose. To gain insights into the regulatory mechanisms of cellulase synthesis, comparative transcriptome analysis was conducted for cultures grown on 2% filter paper, 0.5 and 0.05% cellobiose, and 0.5% birchwood xylan. It was found that cellulose induced a majority of (hemi)cellulases, including 33 cellulases and a cellulosomal scaffoldin (1.3- to 22.7-fold); seven endoxylanases, two mannanases, and two pectatelyases (2- to 16-fold); and pyruvate formate-lyase (PFL, 1.5- to 7-fold). Noticeably, 3- and 2.5-fold increased transcription of a cellobiohydrolase and the cellulosomal scaffoldin precursor were detected in 0.05% than in 0.5% cellobiose. Consistently, 9- and 4-fold higher specific cellobiohydrolase activities were detected in the filter paper and 0.05% cellobiose culture. SDS- and native-PAGE zymograms of cellulose-enriched proteins from the filter paper culture displayed cellulase activities, and cellulolytic "complexes" were enriched from the filter paper- and 0.05% cellobiose-cultures, but not from the 0.5% cellobiose culture. LC-MS/MS identified the cellulosomal scaffoldin precursor in the "complexes" in addition to cellulase, hemicellulase, and PFL proteins. The addition of 0.5% cellobiose, but not 0.05% cellobiose remarkably inhibited strain H1 to degrade filter paper. Therefore, this work reveals a cellobiose-dose related regulatory mechanism of cellulase synthesis by lower for induction and higher for repression, which has extended our understanding of the regulation of microbial cellulase synthesis.

The N-terminal hydrophobic segment of Streptomyces coelicolor FtsY forms a transmembrane structure to stabilize its membrane localization.

FtsY is the receptor of the signal recognition particle that mediates the targeting of integral membrane proteins in bacteria. It was shown that in Escherichia coli, the N-terminal region of FtsY contributes to its interaction with the membrane, but it is not inserted into the membrane. However, this study presents evidence that in Streptomyces coelicolor, FtsY has a hydrophobic region at its N-terminus, which forms a membrane insertion structure and contributes significantly to the binding between FtsY and membrane. Through membrane protein extraction followed by immunoblotting, we demonstrated that deletion of the N-terminal residues 11-39 from the S. coelicolor FtsY (ScFtsY) drastically reduced its membrane-binding capability and that the N-terminus of ScFtsY alone was capable of targeting the soluble EGFP protein onto the membrane with high efficiency. Furthermore, in a labeling experiment with the membrane-impermeable probe Mal-PEG, the ScFtsY N-terminal region was protected by the membrane and was not labeled. This observation indicates that this region was inserted into the membrane.

The pleitropic regulator AdpAch is required for natamycin biosynthesis and morphological differentiation in Streptomyces chattanoogensis.

The complete natamycin (NTM) biosynthetic gene cluster of Streptomyces chattanoogensis was cloned and confirmed by the disruption of pathway-specific activator genes. Comparative cluster analysis with its counterpart in Streptomyces natalensis revealed different cluster architecture between these two clusters. Compared with the highly conserved coding sequences, sequence variations appear to occur frequently in the intergenic regions. The evolutionary change of nucleotide sequence in the intergenic regions has given rise to different transcriptional organizations in the two clusters and resulted in altered gene regulation. These results provide insight into the evolution of antibiotic biosynthetic gene clusters. In addition, we cloned a pleitropic regulator gene, adpA(ch), in S. chattanoogensis. Using the genetic system that we developed for this strain, adpA(ch) was deleted from the genome of S. chattanoogensis. The ΔadpA(ch) mutant showed a conditionally sparse aerial mycelium formation phenotype and defects in sporulation; it also lost the ability to produce NTM and a diffusible yellow pigment normally produced by S. chattanoogensis. RT-PCR analysis revealed that transcription of adpA(ch) was constitutive in YEME liquid medium. By using rapid amplification of 5' complementary DNA ends, two transcription start sites were identified upstream of the adpA(ch) coding region. Quantitative transcriptional analysis showed that the expression level of the NTM regulatory gene scnRI decreased 20-fold in the ΔadpA(ch) mutant strain, while the transcription of the other activator gene scnRII was not significantly affected. Electrophoretic mobility shift assay (EMSA) showed that AdpA(ch) binds to its own promoter but fails to bind to the promoter region of scnRI, indicating that the control of scnRI by AdpA(ch) is exerted in an indirect way. This work not only provides a platform and a new potential target for increasing the titre of NTM by genetic manipulation, but also advances the understanding of the regulation of NTM biosynthesis.

Episodic sitewise positive selection on the signal recognition particle protein Ffh in Actinobacteria.

In this work, we report a case of episodic sitewise positive selection acting on the highly conserved SRP protein Ffh in Actinobacteria. An elevated non-synonymous to synonymous mutation ratio (ω) was observed along the non-terminal branches of the species tree, which contained 11 Actinobacteria species, where positively selected residues were frequently observed within the signal sequence-binding domain. Together with the estimated lineage-specific ω ratios for several core components in the major protein translocation systems, our data suggest that this positive selection might be partially driven by the diversification of signal sequences.