Study on microbial diversity of washing machines.

Health and environmental protection are the development trend of household appliances, coupled with the impact of the COVID-19 epidemic in the past few years. Consumers have unprecedented concerns and expectations about the sterilization and disinfection functions of household appliances. As a washing and nursing equipment for household clothes, the anti-bacterial technology of washing machine has developed rapidly. The new models of washing machines in the market have basically added the function of sterilization. In order to thoroughly solve the problem of sterilization and bacteriostasis of washing machines from the source, the distribution of microbial contamination in washing machines should be fully investigated. At present, there is almost no systematic study on the microbial community structure in washing machines in China. Therefore, the purpose of this study is to analyze the bacterial community structure in Chinese household washing machines. To explore the key factors affecting the bacterial community structure of washing machines. Bacterial communities were comprehensively analyzed by high throughput sequencing. Using chao and shannon indexes as indicators, one-way ANOVA was used to explore the key factors affecting the bacterial community structure of washing machines. A total of 2,882,778 tags and 21,265 OTUs from 522 genera were sequenced from 56 washing machine samples. Genus Mycobacterium, Pseudomonas, Brevundimonas, Sphingomonas, Sphingobium, Enhydrobacter, Methylobacterium, Pseudoxanthomonas, Stenotrophomonas and Sphingopyxis were the top ten bacteria genera in abundance. The effects of sources, types, frequency of utilization, sampling locations and service life of washing machines on bacterial diversity in washing machine were systematically analyzed. The statistical analysis showed that service life was an important factor affecting bacterial diversity in washing machine. Our study lays a foundation for directional screening of characteristic microorganisms with targeted characters including malodor-producing, fouling, pathogenic and stress-resistance, the antibacterial evaluation, metabolic mechanism of key characteristic microorganisms as well as antibacterial materials development. At present, the sterilization technology of washing machines has not been fully in combination with the distribution survey of microorganisms in washing machines. According to the specific microorganism distribution condition of the washing machine, the key distribution positions and the types of specific microorganisms contained in different positions, conduct more targeted sterilization treatment. This will help to completely solve the problem of microbial growth in washing machines from the source.

Mechanism of TonB-dependent transport system in Halomonas alkalicola CICC 11012s in response to alkaline stress.

Halomonas alkalicola CICC 11012s can grow at pH 12.5, the highest pH at which the organisms in the genus Halomonas can grow. Genomic analysis reveals that H. alkalicola adapts to alkaline stress using a variety of adaptive strategies; however, the detailed mechanism for its growth at high-alkaline conditions has not been elucidated. Therefore, in this study, the adaptations of H. alkalicola in response to extreme alkaline stress were investigated. A sharp decrease of alkaliphilic tolerance was observed in mutants E. coli ΔEctonB and H. alkalicola ΔHatonB. Expressions of the gene clusters encoding TonB-dependent transport system and iron complex transport system in H. alkalicola grown under extreme alkaline conditions were markedly up-regulated. We then compared the intracellular ionic iron content and iron-chelating ability of mutant strain with those of wild-type strain to understand the influence of TonB-dependent transport system on the alkaline responses. The results indicated that the presence of TonB-dependent transport system increased the alkaline tolerance of H. alkalicola grown at high-alkaline conditions, but had no effects when the strain was grown at neutral pH and low-alkaline conditions. Meanwhile, the presence of this system increased the transport and accumulation of ionic irons to maintain intracellular metabolic homeostasis, which in turn could increase the tolerance of the strain to extreme alkaline conditions. Based on the results, we established a model representing the interactions between TonB-dependent transport system, alkaline tolerance, and intracellular ionic iron that could help deepen the understanding of the alkaline response mechanism of alkaliphilic bacteria.

Bacillus aquiflavi sp. nov., isolated from yellow water of strongly flavored Chinese baijiu.

A Gram-stain-positive, strictly aerobic and rod-shaped bacterium, designated as 3 H-10T, was isolated from a yellow water sample collected from the manufacturing process of strong flavor Chinese baijiu in Yibin region of Sichuan province (PR China). Oval endospores were formed at the subtermini of cells with swollen sporangia. The isolate was able to grow at temperatures of 20-45 °C (optimum growth at 37 °C), at pH 6.0-10.0 (optimum growth at pH 8.0) and in the presence of 0-2 % (w/v) NaCl (optimum growth with 0 % NaCl). Ribose was the major cell-wall sugar, and meso-diaminopimelic acid (meso-DAP) was the diagnostic amino acid. The main polar lipids of 3 H-10T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). MK-7 was predominant menaquinone and iso-C15 : 0 (60.7 %) was the major fatty acid. Comparisons of 16S rRNA gene sequence indicated that 3 H-10T was most closely related to Bacillus mesophilus SA4T (96.30 %), Bacillus ginsengihumi Gsoil 114T (96.27 %) and Bacillus shackletonii LMG 18435T (96.27 %). The average nucleotide identity (ANI) values between strain 3 H-10T and the three type strains mentioned above were 69.56, 70.19 and 70.67 %, respectively. The genomic DNA G+C content was 35.4 mol%. On the basis of its phenotypic, chemotaxonomic and phylogenetic properties, strain 3 H-10T represents a novel species of the genus Bacillus, for which the name Bacillus aquiflavi sp. nov. is proposed. The type strain is Bacillus aquiflavi 3 H-10T (=CICC 24755T=JCM 33703T).

Paenibacillus maysiensis sp. nov., a Nitrogen-Fixing Species Isolated from the Rhizosphere Soil of Maize.

A novel bacterium SX-49T with nitrogen-fixing capability was isolated from the rhizosphere soil of maize. Phylogenetic analysis of nifH gene fragment and 16S rRNA gene sequence revealed that the strain SX-49T is a member of the genus Paenibacillus. Values of 16S rRNA gene sequence similarity were highest between SX-49T and P. jamilae DSM 13815T (97.0%), P. brasiliensis DSM 14914T (97.8%), P. polymyxa DSM 36T (97.5%), and P. terrae DSM 15891T (98.8%). The similarity between SX-49T and other Paenibacillus species was < 97.0%. DNA-DNA hybridization values between strain SX-49T and the four type strains were P. jamilae DSM 13815T: 40.6%, P. brasiliensis DSM 14914T: 27.9%, P. polymyxa DSM 36T: 29.2%, and P. terrae DSM 15891T: 66.4%. The DNA G+C content of SX-49T was 46.4 mol%. The predominant fatty acids were anteiso-C15:0, C16:0 and iso-C16:0. The predominant isoprenoid quinone was MK-7. The genome contains 5628 putative protein-coding sequences (CDS), 6 rRNAs and 56 tRNAs. The phenotypic and genotypic characteristics, DNA-DNA relatedness, and genome features suggest that SX-49T represents a novel species of the genus Paenibacillus, and the name Paenibacillus maysiensis sp. nov. is proposed.

Genome sequencing and heterologous expression of antiporters reveal alkaline response mechanisms of Halomonas alkalicola.

Halomonas alkalicola CICC 11012s is an alkaliphilic and halotolerant bacterium isolated from a soap-making tank (pH > 10) from a household-product plant. This strain can propagate at pH 12.5, which is fatal to most bacteria. Genomic analysis revealed that the genome size was 3,511,738 bp and contained 3295 protein-coding genes, including a complete cell wall and plasma membrane lipid biosynthesis pathway. Furthermore, four putative Na+/H+ and K+/H+ antiporter genes, or gene clusters, designated as HaNhaD, HaNhaP, HaMrp and HaPha, were identified within the genome. Heterologous expression of these genes in antiporter-deficient Escherichia coli indicated that HaNhaD, an Na+/H+ antiporter, played a dominant role in Na+ tolerance and pH homeostasis in acidic, neutral and alkaline environments. In addition, HaMrp exhibited Na+ tolerance; however, it functioned mainly in alkaline conditions. Both HaNhaP and HaPha were identified as K+/H+ antiporters that played an important role in high alkalinity and salinity. In summary, genome analysis and heterologous expression experiments demonstrated that a complete set of adaptive strategies have been developed by the double extremophilic strain CICC 11012s in response to alkalinity and salinity. Specifically, four antiporters exhibiting different physiological roles for different situations worked together to support the strain in harsh surroundings.

Comparative genomic analysis of N2-fixing and non-N2-fixing Paenibacillus spp.: organization, evolution and expression of the nitrogen fixation genes.

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N2-fixing strains. The heterogeneity of the 31 genomes (15 N2-fixing and 16 non-N2-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N2-fixing strains. The nif cluster is under control of a σ(70)-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe-S] cluster is highly conserved in N2-fixing and non-N2-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N2-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N2-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N2-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.