Ximing Chen

Optimization of electroporation conditions for Arthrobacter with plasmid PART2

A prerequisite for genetic studies of Arthrobacter is a high efficiency transformation system that allows for DNA transfer, transposon mutagenesis, and expression of specific genes. In this study, we develop a detailed electroporation method through a systematic examination of the factors involved in the entire electroporation process. Key features of this procedure, including the addition of penicillin to cells during the early log phase of growth and the presence of 0.5M sorbitol in the electroporation and recovery media, produced the greatest increases in transformation efficiency and consistency of results. The transformation rate also varied depending on the electrical parameters, DNA concentration, and recovery time period. Using optimum conditions, we generally achieved an efficiency of 6.8 × 10(7) transformants per microgram of PART2 for Arthrobacter sp. A3. This protocol was also successfully applied to other Arthrobacter species. Therefore, we conclude that the proposed method is rapid, simple and convenient, which allows a transformation trial to be accomplished in minutes.

Regulation of expression of trehalose-6-phosphate synthase during cold shock in Arthrobacter strain A3

Trehalose is a chemical chaperone known to protect a variety of organisms against cold stress. Members of the genus Arthrobacter, which belongs to the Actinomycetales group, exhibit strong resistance to stress conditions, but exactly how trehalose synthesis is regulated in conditions of cold stress is still unknown. Here, we report that Arthrobacter strain A3, which was isolated from the alpine permafrost, has only two trehalose synthesis pathways (OtsA/B and TreS), while other Arthrobacter spp. have three. Mutants and immunoblot analyses indicate that trehalose is mainly synthesized via OtsA at low temperatures in Arthrobacter strain A3. Therefore, we have focused on the regulation of OtsA expression during cold shock. The results indicated that both low temperature and accumulation of trehalose can inhibit OtsA expression. The elongation factor Tu, which binds to OtsA, stabilizes the expression of OtsA in the cold.

The diversity and biogeography of the communities of Actinobacteria in the forelands of glaciers at a continental scale

Glacier forelands, where the initially exposed area is unvegetated with minimal human influence, are an ideal place for research on the distributions and biogeography of microbial communities. Actinobacteria produce many bioactive substances and have important roles in soil development and biogeochemical cycling. However, little is known about the distribution and biogeography of Actinobacteria in glacier forelands. Therefore, we investigated the patterns of diversity and the biogeography of actinobacterial communities of the inhabited forefields of 5 glaciers in China. Of the bacteria, the mean relative abundance of Actinobacteria was 13.1%, and 6 classes were identified in the phylum Actinobacteria. The dominant class was Actinobacteria (57%), which was followed in abundance by Acidimicrobiia (19%) and Thermoleophilia (19%). When combined, the relative abundance of the other three classes, the MB-A2-108, Nitriliruptoria and Rubrobacteria, was only 2.4%. Abiogeographic pattern in the forelands of the 5 glaciers in China was not detected for actinobacterial communities. Compared with 7 other actinobacterial communities found in the forelands of glaciers globally, those in the Southern Hemisphere were significantly different from those in the Northern Hemisphere. Moreover, the communities were significantly different on the separate continents of the Northern Hemisphere. The dissimilarity of the actinobacterial communities increased with geographic distance (r=0.428, p=0.0003). Because of environmental factors, the effect of geography was clear when the distance exceeded a certain continent-level threshold. With the analysis of indicator species, we found that each genus had a geographic characteristic, which could explain why the communities with greater diversity were more strongly affected by biogeography.

Evolution of Threonine Aldolases, a Diverse Family Involved in the Second Pathway of Glycine Biosynthesis

Threonine aldolases (TAs) catalyze the interconversion of threonine and glycine plus acetaldehyde in a pyridoxal phosphate-dependent manner. This class of enzymes complements the primary glycine biosynthetic pathway catalyzed by serine hydroxymethyltransferase (SHMT), and was shown to be necessary for yeast glycine auxotrophy. Because the reverse reaction of TA involves carbon–carbon bond formation, resulting in a β-hydroxyl-α-amino acid with two adjacent chiral centers, TAs are of high interests in synthetic chemistry and bioengineering studies. Here, we report systematic phylogenetic analysis of TAs. Our results demonstrated that l-TAs and d-TAs that are specific for l- and d-threonine, respectively, are two phylogenetically unique families, and both enzymes are different from their closely related enzymes SHMTs and bacterial alanine racemases (ARs). Interestingly, l-TAs can be further grouped into two evolutionarily distinct families, which share low sequence similarity with each other but likely possess the same structural fold, suggesting a convergent evolution of these enzymes. The first l-TA family contains enzymes of both prokaryotic and eukaryotic origins, and is related to fungal ARs, whereas the second contains only prokaryotic l-TAs. Furthermore, we show that horizontal gene transfer may occur frequently during the evolution of both l-TA families. Our results indicate the complex, dynamic, and convergent evolution process of TAs and suggest an updated classification scheme for l-TAs.

The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures

The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Here, we cloned the otsA gene from Arthrobacter sp. Cjts, and expressed and then purified the recombinant proteins. Enzyme activity analysis indicated that the high catalytic efficiency of OtsA from Arthrobacter sp. Cjts resulted from the high affinity of the enzyme for uridine 5′-diphosphoglucose (UDP-Glc) at low temperatures. We also confirmed that the N-loop sequence of OtsA has a large effect on its affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be directly related to the catalytic efficiency of OtsA at low temperatures.

Effects of enhanced atmospheric ammonia on photosynthetic characteristics of two maize (Zea mays L.) cultivars with various nitrogen supply across long-term growth period and their diurnal change patterns

We investigated the effect of enhanced atmospheric ammonia (NH3) in combination with low and high nitrogen (LN and HN, respectively) growth medium on photosynthetic characteristics of two maize (Zea mays L.) cultivars (NE5 with high- and SD19 with low N-use efficiency) across long-term growth period and their diurnal change patterns exposed to 10 nl l−1 and 1,000 nl l−1 NH3 fumigation in open-top chambers (OTCs). Regardless of the level of N in medium, increased NH3 concentration promoted maximum net photosynthetic rate (Pmax) and apparent quantum yield (AQY) of both cultivars at earlier growth stages, but inhibited Pmax of NE5 from silking to maturity stage and that of SD19 at maturity stage only above the ambient concentration. Greater positive/less negative responses were predominant in the LN than in the HN treatment, especially for SD19. Dark respiration rate (RD) remained more enhanced in the LN than in the HN treatment for SD19 as well as increased in the LN while decreased in the HN treatment for NE5 at their silking stage, following exposure to elevated NH3 concentration. Additionally, enhanced atmospheric NH3 increased net photosynthetic rate (PN) and stomatal conductance (gs) but reduced intercellular CO2 concentration (Ci) of both cultivars with either the LN or HN treatment during the diurnal period at tasseling stage. The diurnal change patterns of PN and gs showed bimodal curve type and those of Ci presented single W-curve type for NE5, when NH3 concentration was enhanced. As for SD19, single-peak curve type was showed for both PN and gs while single V-curve type for Ci. All results supported the hypothesis that appropriately enhanced atmospheric NH3 can increase assimilation of CO2 by improving photosynthesis of maize plant, especially at earlier growth stages and after photosynthetic “noon-break” point. These impacts of elevated NH3 concentration were more beneficial for SD19 as compared to those for NE5, especially in the LN supply environment.

Complete genome sequence of a psychotrophic Arthrobacter strain A3 (CGMCC 1.8987), a novel long-chain hydrocarbons producer

Arthrobacter strain A3, a psychotrophic bacterium isolated from the Tian Shan Mountain of China, can degrade the cellulose and synthesis the long-chain hydrocarbons efficiently in low temperature. Here we report the complete genome sequence of this bacterium. The complete genome sequence of Arthrobacter strain A3, consisting of a cycle chromosome with a size of 4.26 Mbp and a cycle plasmid with a size of 194kbp. In this genome, a hydrocarbon biosynthesis gene cluster (oleA, oleB/oleC and oleD) was identified. To resistant the extreme environment, this strain contains a unique mycothiol-biosynthetic pathway (mshA-D), which has not been found in other Arthrobacter species before. The availability of this genome sequence allows us to investigate the genetic basis of adaptation to growth in a nutrient-poor permafrost environment and to evaluate of the biofuel-synthetic potential of this species.

Genome Sequence of Streptomyces violaceusniger Strain SPC6, a Halotolerant Streptomycete That Exhibits Rapid Growth and Development

ABSTRACT Streptomyces violaceusniger strain SPC6 is a halotolerant streptomycete isolated from the Linze desert in China. The strain has a very high growth rate and a short life cycle for a streptomycete. For surface-grown cultures, the period from spore germination to formation of colonies with mature spore chains is only 2 days at 37°C. Additionally, the strain is remarkably resistant to osmotic, heat, and UV stress compared with other streptomycetes. Analysis of the draft genome sequence indicates that the strain has the smallest reported genome (6.4 Mb) of any streptomycete. The availability of this genome sequence allows us to investigate the genetic basis of adaptation for growth in an extremely arid environment.

Diversity and Succession of Actinobacteria in the Forelands of the Tianshan Glacier, China

ABSTRACT Actinobacteria are typically soil bacteria that have important roles in soil development and biogeochemical cycling. However, little is known about the occurrence or the succession of communities of Actinobacteria in new habitats. In this study, we investigated the diversity and succession of the actinobacterial communities that inhabited the forelands of the Tianshan Glacier (China), which ranged in successional age from 0 to 100 years since the forefield was deglaciated. Actinobacteria was one of the dominant phyla in the glacier foreland and included the orders Acidimicrobiales, Actinomycetales, Rubrobacteriales and Solirubrobacteriales. Actinomycetales was the dominant order, but its relative abundance decreased through the chronosequence. Acidimicrobiales and Solirubrobacteriales were more abundant in the late stages of succession than in the early ones. The abundance of Rubrobacteriales was only high at 74a. The dominant genera Nocardioides and Arthrobacter were widely distributed and were found in each stage of succession. With nonparametric and rarefaction estimated analyses, we found that the phylotype richness of Actinobacteria was significantly correlated with time (r = 0.886, p = 0.019). The succession of actinobacterial communities was divided into 3 stages: the early stage (6a), the intermediate stage (10a and 20a) and the late stage (60a, 74a, and 100a). Based on the canonical correspondence analysis, the actinobacterial communities were affected significantly by soil pH (r = −0.834, p = 0.039) and somewhat by the C/N ratio (r = 0.783, p = 0.066). The nonmetric multidimensional scaling analysis showed that the effect of geographical isolation on the actinobacterial communities was greater than that of the soils in the development of the chronosequence.

A trehalose biosynthetic enzyme doubles as an osmotic stress sensor to regulate bacterial morphogenesis

The dissacharide trehalose is an important intracellular osmoprotectant and the OtsA/B pathway is the principal pathway for trehalose biosynthesis in a wide range of bacterial species. Scaffolding proteins and other cytoskeletal elements play an essential role in morphogenetic processes in bacteria. Here we describe how OtsA, in addition to its role in trehalose biosynthesis, functions as an osmotic stress sensor to regulate cell morphology in Arthrobacter strain A3. In response to osmotic stress, this and other Arthrobacter species undergo a transition from bacillary to myceloid growth. An otsA null mutant exhibits constitutive myceloid growth. Osmotic stress leads to a depletion of trehalose-6-phosphate, the product of the OtsA enzyme, and experimental depletion of this metabolite also leads to constitutive myceloid growth independent of OtsA function. In vitro analyses indicate that OtsA can self-assemble into protein networks, promoted by trehalose-6-phosphate, a property that is not shared by the equivalent enzyme from E. coli, despite the latter’s enzymatic activity when expressed in Arthrobacter. This, and the localization of the protein in non-stressed cells at the mid-cell and poles, indicates that OtsA from Arthrobacter likely functions as a cytoskeletal element regulating cell morphology. Recruiting a biosynthetic enzyme for this morphogenetic function represents an intriguing adaptation in bacteria that can survive in extreme environments.