Proteus appendicitidis sp. nov., isolated from the appendiceal pus of an appendicitis patient in Yongzhou, China.

A Gram-negative, facultatively anaerobic, short rod-shaped bacterium, designated as strain HZ0627T, was isolated from the appendiceal pus of a patient with appendicitis in Yongzhou, Hunan, China. This strain was subjected to comprehensive phenotypic, phylogenetic, and genomic analyses using polyphasic taxonomic methods. Phylogenetic analysis of the 16S rRNA gene sequence revealed that this strain belonged to the genus Proteus and the family Morganellaceae, whereas that based on the rpoB gene sequence and phylogenomic analysis demonstrated that this strain was distinctly separated from other type strains of Proteus species. Moreover, whole-genome-based analyses, including in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI), revealed that strain HZ0627T had much lower isDDH rates (24.5-55.6%) and ANI (82.04-93.90%) than those of the thresholds (i.e., 70% and 95%, respectively) for species delineation, when compared to the type strains of other Proteus species. The cellular fatty acid profile of strain HZ0627T was dominated by C16:0 (34.5%), cyclo C17:0 (25.8%), C14:0 (12.6%), C16:1 iso I/14:0 3-OH (7.7%), C18:1ω7c/18:1ω6c (6.5%), and C16:1ω7c/16:1ω6c (4.9%), which clearly differentiated it from the documented type strains of Proteus species. In addition, several specific physiological traits, including optimal growth temperature, tolerance to sodium chloride, and carbon source utilization, differed from those of other Proteus species. Therefore, we propose the name Proteus appendicitidis sp. nov. for strain HZ0627T (= CCTCC AB 2022380T = KCTC 92986T), which represents the type strain of this novel Proteus species.

Multiple transitions between realms shape relict lineages of Proteus cave salamanders.

In comparison to biodiversity on Earth's surface, subterranean biodiversity has largely remained concealed. The olm (Proteus anguinus) is one of the most enigmatic extant cave inhabitants, and until now little was known regarding its genetic structure and evolutionary history. Olms inhabit subterranean waters throughout the Dinaric Karst of the western Balkans, with a seemingly uniform phenotypic appearance of cave-specialized traits: an elongate body, snout and limbs, degenerated eyes and loss of pigmentation ("white olm"). Only a single small region in southeastern Slovenia harbours olms with a phenotype typical of surface animals: pigmented skin, eyes, a blunt snout and short limbs ("black olm"). We used a combination of mitochondrial DNA and genome-wide single nucleotide polymorphism data to investigate the molecular diversity, evolutionary history and biogeography of olms along the Dinaric Karst. We found nine deeply divergent species-level lineages that separated between 17 and 4 million years ago, while molecular diversity within lineages was low. We detected no signal of recent admixture between lineages and only limited historical gene flow. Biogeographically, the contemporaneous distribution of lineages mostly mirrors hydrologically separated subterranean environments, while the historical separation of olm lineages follows microtectonic and climatic changes in the area. The reconstructed phylogeny suggests at least four independent transitions to the cave phenotype. Two of the species-level lineages have miniscule ranges and may represent Europe's rarest amphibians. Their rarity and the decline in other lineages call for protection of their subterranean habitats.

Integrative conjugative elements mediate the high prevalence of tmexCD3-toprJ1b in Proteus spp. of animal source.

IMPORTANCE: The emergence and spread of tmexCD-toprJ have greatly weakened the function of tigecycline. Although studies have demonstrated the significance of Proteus as carriers for tmexCD-toprJ, the epidemic mechanism and characteristics of tmexCD-toprJ in Proteus remain unclear. Herein, we deciphered that the umuC gene in VRIII of SXT/R391 ICEs was a hotspot for the integration of tmexCD3-toprJ1b-bearing mobile genetic elements by genomic analysis. The mobilization and dissemination of tmexCD3-toprJ1b in Proteus were mediated by highly prevalent ICEs. Furthermore, the co-occurrence of tmexCD3-toprJ1b-bearing ICEs with other chromosomally encoded multidrug resistance gene islands warned that the chromosomes of Proteus are significant reservoirs of ARGs. Overall, our results provide significant insights for the prevention and control of tmexCD3-toprJ1b in Proteus.

Complicated pyelonephritis caused by Proteus alimentorum in a woman with peritoneal cancer: a case report.

BACKGROUND: Proteus spp. are widespread in the environment and comprise a part of the normal flora of the human gastrointestinal tract. Only six species in this genus, including Proteus mirabilis, Proteus vulgaris, Proteus terrae, Proteus penneri, Proteus hauseri, and Proteus faecis, have been isolated from human clinical specimens. However, there are no reports of Proteus alimentorum isolated from humans, and the clinical characteristics of P. alimentorum infection are unknown. CASE PRESENTATION: An 85-year-old female patient with peritoneal cancer was hospitalized for complicated pyelonephritis and bacteremia caused by P. alimentorum. The patient received antimicrobial therapy and was discharged on day 7 of hospitalization. No recurrence was observed 14 days after the treatment. Various methods were used to identify the Proteus sp. Furthermore, the VITEK-2 GN ID card resulted in low discrimination between P. hauseri and P. penneri. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed P. hauseri with a spectral score of 2.22 as the best match. Nevertheless, the pathogen was identified as P. alimentorum based on genetic investigation using 16 S rRNA gene sequencing and biochemical tests. CONCLUSION: Proteus alimentorum is a human pathogen, and its infection has an excellent therapeutic response to antimicrobials based on antimicrobial susceptibility. Genomic methods may be helpful for the precise identification of P. alimentorum.

Characterization, genome analysis and antibiofilm efficacy of lytic Proteus phages RP6 and RP7 isolated from university hospital sewage.

The crystalline formation of biofilms by Proteus blocks the urine flow which often complicates the health care of catheterized patients. Bacteriophages has been highlighted as a promising tool to control biofilm-mediated bacterial infections. Here, we isolated and characterized two newly isolated lytic phages capable of infecting clinical isolates of P. mirabilis and P. vulgaris. Moreover, insights regarding the biological and molecular characterization were analysed. Both RP6 and RP7 phages showed a Proteus-genus-specific profile, administering no lytic activity against other family of Enterobacteriaceae. The optimal MOI value of the RP6 and RP7 phages were determined as 0.1 and 0.01, respectively. The one-step growth curve showed that RP6 and RP7 phages have a short latent period of 20 min and large burst size of 220-371 PFU/ML per infected host cell. Bacteria growth was reduced immediately after the phages were added, which is shown by the optical density (OD) measurement after 24 hr. Proteus phage RP6 and RP7 were found to eradicate both the planktonic and mature biofilms produced by the Proteus isolates tested. Genome sequence of Proteus phage RP6 was found to be 58,619 bp, and a G-C content of 47%. Also, Proteus phage RP7 genome size was 103,593 bp with G-C ratio of 38.45%. A total of 70 and 172 open reading frame (ORF) was encoded in RP6 and RP7 phage genomes, respectively. Interestingly, there were no tRNA encoded by Proteus phage RP6 genome even though there is a significant G-C content difference between the phage and its host. Additionally, the exhibition of highly lytic activity and absence of virulence and antibiotic-resistant genes in both Proteus RP6 and RP7 phages emphasized that this newly isolated phages are promising for potential therapeutic phages.

Frequency distribution of virulence factors and antibiotic resistance genes in uropathogenic Proteus species isolated from clinical samples.

One of the most common causes of urinary tract infections (UTIs) is Proteus species. Because there is little information on the pathogenicity of Proteus species isolated from Iran, we assessed their virulence characteristics and antibiotic resistance in this study. In Shahrekord, Iran, 260 isolates of Proteus causing UTIs were identified from patients. Polymerase chain reaction for gene amplification was used to determine virulence features and antibiotic resistance gene distribution in uropathogenic Proteus spp. After biochemical and molecular analysis, 72 (27.69%) of the 260 collected samples were recognized as Proteus mirabilis, and 127 (48.84%) specimens were Pr. vulgaris in both male and female forms. A significant interaction effect between Pr. mirabilis and Pr. vulgaris infections and the sex of patients was seen in both the male and female groups. No statistically significant difference was observed between Pr. mirabilis infection and season in different year seasons. However, in different seasons of the year, a statistically significant difference was observed between infection with Pr. vulgaris in autumn and other seasons. There was a considerable difference between Pr. mirabilis and Pr. vulgaris infections at different ages in various age groups. As people aged, infections occurred more frequently. Fim,pap,kspMT, and set1 genes had the highest expression in both Pr. vulgaris and Pr. mirabilis. Also, the highest rate of antibiotic resistance of Pr. vulgaris and Pr. mirabilis is attributed to the high expression of aac(3)-IV,tet(A), and blaSHV genes. In conclusion, identifying these genes as the key controllers of Proteus virulence factors might help with better infection management.

Molecular Analysis of Virulence Genes HpmB and rsbA among proteus Species Isolated from Different Infectious Cases in Iraq.

Proteus species (spp.) is considered one of the widely spread pathogens worldwide. Proteus spp. can be detected in contaminated water, soil, and manure, aiding the decomposition of organic substances from animals. Proteus is a gram-negative bacterium that causes a wide range of human illnesses. This study aimed to find some virulence genes in Proteus spp. from different sources, including the laboratories of government hospitals in Karbala, Al-Hussies, and Al-Muthanna, Iraq. Fifty swab samples were collected from patients' wounds, ears, and sputum. Clinicians collected swab samples for identification. In total, 17 sputum samples, 13 ear samples, and 20 wound samples were collected from 27 (54%) females and 23 (46%) males. The virulence genes hpmB and rsbA were identified after the genomic diagnosis of Proteus spp. Thirteen Proteus isolates were identified using the hpmB primer, and 16 isolates were identified using the rsbA primer. The DNA sequence analysis of rsbA and hpmB genes revealed that all samples shared 99.52% identity for the rsbA gene, whereas the hpmB gene differed from one sample to the next. The sequence results are available at the NCBI under the accession numbers (LC661938) and (LC661939), respectively.

Evaluation of antibiotic resistance in Proteus spp: a growing trend that worries Public Health. Results of 10 Years of Analysis.

The genus Proteus includes several species among which Proteus mirabilis is by far the most commonly detected in clinical specimens. In the last twenty years, isolates with multiple acquired resistance genes have been detected, especially in the hospital environment, with a significant impact on the treatment of infections. This research is a ten-year cross-sectional study reporting the detection rates and the antibiotic susceptibility of Proteus spp. in clinical specimens from a healthcare setting in Southern Italy. Of all the 1,600 clinical samples sent to the laboratory, 4.4% were positive to Proteus spp., with P. mirabilis by far the most detected one (83.1%), especially in lower limb ulcers and urines. Moreover, we noted a significant increase of 1200% in the detection rate from 2011 to 2020. Finally, we reported a significant and constantly increasing trend in the detection of antibiotic-resistant strains, ranging from 48.4% in 2011 to 74% in 2020. Our results highlight a clear and significant increase in Proteus spp. detection in a typical hospital setting with a parallel increase in the detection of antibiotic-resistant strains. Therefore, Proteus spp. can be considered one of the main emerging pathogenic bacteria in the hospital environment.

Identification of Genes Encoding Aminoglycoside Modifying Enzymes among Clinical Isolates of Proteus species at a Tertiary Care Hospital in Dhaka, Bangladesh.

Proteus is considered as one of the major opportunistic pathogens liable for nosocomial infections and acquired several resistances to a wide range of antimicrobials such as aminoglycosides. The most common mechanism of aminoglycoside resistance is the inactivation of drugs by modifying enzymes. So, this cross-sectional study was undertaken to investigate the occurrence of aminoglycoside resistance and identify aminoglycoside modifying enzyme (AME) genes among clinical isolates of aminoglycoside resistant Proteus spp. A total of 40 Proteusmirabilis and Proteus vulgaris were isolated in the Department of Microbiology of Dhaka Medical College, Dhaka, Bangladesh from July 2018 to June 2019 of 500 wound swab & pus, urine and blood samples. Disk diffusion test was performed by modified Kirby Bauer method. Minimum inhibitory concentration (MIC) of amikacin was determined by agar dilution method. PCR was used to detect aac(3)-Ia, aac(6')-Ib, ant(4')-IIa, ant(2'')-Ia a and aph(3'')-Ib AMEs genes among aminoglycoside resistant Proteus spp. Sequencing of aac(6')-Ib gene was performed to identify aac(6')-Ib-cr variant. Thirty-two (80%) aminoglycoside resistant isolates were detected during disk-diffusion technique. The marked increase in MIC was observed between 256 - ≥2048µg/ml to amikacin. The most prevalent AME-genes were aac(6')-Ib (37.5%), ant(2'')-Iaa (21.86) followed by ant(4')-IIa(12.5%), aph(3'')-Ib (12.5%) andaac(3)-Ia (9.38%). The most frequent combination was aac(6')-Ib + aac(3)-Ia+ant(2'')-Iaa and aac(6')-Ib + ant(4')-IIa + aph(3'')-Ib(2 strains) followed by aac(6')-Ib + aac(3)-Ia(1 strain). Sequencing of aac(6')-Ib gene in this study did not harbor aac(6')-Ib-cr variant gene. The results of this study provide insight into the presence of high AME-genes among Proteus spp. in Bangladesh.

Quick Identification and Differentiation of Proteus spp. by PCR and RFLP Along with Their Antibiotic Resistance Pattern.

Different Proteus species are encountered in human infections and may vary with the type of infections they cause. So, the present study was conducted to detect species of Proteus by PCR and RFLP along with their antibiotic resistance pattern. This cross-sectional study was conducted in the Department of Microbiology of Dhaka Medical College, Dhaka, Bangladesh, from July 2018 to June 2019. A total of 500 wound swab and pus, urine and blood samples were tested for bacterial pathogens. Proteus spp. were identified and differentiated by biochemical test, PCR and RFLP. Antibiotic susceptibility was performed by disc-diffusion technique. Fourty Proteus spp. was isolated from 300 culture positive samples, giving 13.33% prevalence of Proteus infections. Proteus mirabilis and Proteus vulgaris were identified by culture, biochemical test, PCR and RFLP. The results were similar by both methods (biochemical tests and PCR). RFLP of 16S rRNA fragments digested with HaeIII revealed that P. mirabilis consisted of two bands at approximately 110 and 190 bp and P. vulgaris consisted of three bands at approximately 100, 180 and 220 bp. The proportion (80%) of P. mirabilis was more than P. vulgaris. Highest proportion (77.5%) of Proteus spp. was isolated from wound swab and pus followed by urine samples. A significant proportion of Proteus spp. was multidrug resistant (90%) and extensively drug resistant (37.5%). Fosfomycin was found the most sensitive drug followed by imipenem. This study provided an insight into antibiotic resistance pattern of Proteus spp. and showed high level resistance towards commonly used antimicrobial agents. PCR and RFLP may be suitable method to identify and differentiate species of Proteus and to treat them accordingly.

Multilocus sequence analysis for the taxonomic updating and identification of the genus Proteus and reclassification of Proteus genospecies 5 O'Hara et al. 2000, Proteus cibarius Hyun et al. 2016 as later heterotypic synonyms of Proteus terrae Behrendt et al. 2015.

BACKGROUND: Members of the genus Proteus are mostly opportunistic pathogens that cause a variety of infections in humans. The molecular evolutionary characteristics and genetic relationships among Proteus species have not been elucidated to date. In this study, we developed a multilocus sequence analysis (MLSA) approach based on five housekeeping genes (HKGs) to delineate phylogenetic relationships of species within the genus Proteus. RESULTS: Of all 223 Proteus strains collected in the current study, the phylogenetic tree of five concatenated HKGs (dnaJ, mdh, pyrC, recA and rpoD) divided 223 strains into eleven clusters, which were representative of 11 species of Proteus. Meanwhile, the phylogenetic trees of the five individual HKGs also corresponded to that of the concatenated tree, except for recA, which clustered four strains at an independent cluster. The evaluation of inter- and intraspecies distances of HKG concatenation indicated that all interspecies distances were significantly different from intraspecies distances, which revealed that these HKG concatenations can be used as gene markers to distinguish different Proteus species. Further web-based DNA-DNA hybridization estimated by genome of type strains confirmed the validity of the MLSA, and each of eleven clusters was congruent with the most abundant Proteus species. In addition, we used the established MLSA method to identify the randomly collected Proteus and found that P. mirabilis is the most abundant species. However, the second most abundant species is P. terrae but not P. vulgaris. Combined with the genetic, genomic and phenotypic characteristics, these findings indicate that three species, P. terrae, P. cibarius and Proteus genospecies 5, should be regarded as heterotypic synonyms, and the species should be renamed P. terrae, while Proteus genospecies 5 has not been named to date. CONCLUSIONS: This study suggested that MLSA is a powerful method for the discrimination and classification of Proteus at the species level. The MLSA scheme provides a rapid and inexpensive means of identifying Proteus strains. The identification of Proteus species determined by the MLSA approach plays an important role in the clinical diagnosis and treatment of Proteus infection.

Significance and roles of synonymous codon usage in the evolutionary process of Proteus.

Proteus spp. bacteria frequently serve as opportunistic pathogens that can infect many animals and show positive survival and existence in various natural environments. The evolutionary pattern of Proteus spp. is an unknown topic, which benefits understanding the different evolutionary dynamics for excellent bacterial adaptation to various environments. Here, the eight whole genomes of different Proteus species were analyzed for the interplay between nucleotide usage and synonymous codon usage. Although the orthologous average nucleotide identity and average nucleotide identity display the genetic diversity of these Proteus species at the genome level, the principal component analysis further shows that these species sustain the specific genetic niche at the aspect of synonymous codon usage patterns. Interestingly, although these Proteus species have A/T rich genes with underrepresented G (guanine) or C (cytosine) at the third codon positions and overrepresented A or T at these positions, some synonymous codons with A or T end are obviously suppressed in usage. The overall codon usage pattern reflected by the effective number of codons (ENC) has a significantly positive correlation with GC3 content (GC content at the third codon position), and ENC has a significantly negative correlation with the adaptation index for these species. These results suggest that the mutation pressure caused by nucleotide composition constraint serves as a dominant evolutionary dynamic driving evolutionary trend of Proteus spp., along with other selections related to natural selection, replication and fine-tune translation, and so on. Taken together, the analyses help to understand the evolutionary interplay between nucleotide and codon usage at the gene level of Proteus.

Comparative genomic analysis of Proteus spp. isolated from tree shrews indicated unexpectedly high genetic diversity.

Proteus spp. are commensal gastrointestinal bacteria in many hosts, but information regarding the mutual relationships between these bacteria and their hosts is limited. The tree shrew is an alternative laboratory animal widely used for human disease research. However, little is known about the relationship between Proteus spp. and tree shrews. In this study, the complete genome sequencing method was used to analyse the characteristics of Proteus spp. isolated from tree shrews, and comparative genomic analysis was performed to reveal their relationships. The results showed that 36 Proteus spp. bacteria were isolated, including 34 Proteus mirabilis strains and two Proteus vulgaris strains. The effective rate of sequencing was 93.53%±2.73%, with an average GC content of 39.94%±0.25%. Briefly, 3682.89±90.37, 2771.36±36.01 and 2832.06±42.49 genes were annotated in the NCBI non-redundant nucleotide database (NR), SwissProt database and KEGG database, respectively. The high proportions of macrolide-, vancomycin-, bacitracin-, and tetracycline-resistance profiles of the strains were annotated in the Antibiotic Resistance Genes Database (ARDB). Flagella, lipooligosaccharides, type 1 fimbriae and P fimbriae were the most abundantly annotated virulence factors in the Virulence Factor Database (VFDB). SNP variants indicated high proportions of base transitions (Ts), homozygous mutations (Hom) and non-synonymous mutations (Non-Syn) in Proteus spp. (P<0.05). Phylogenetic analysis of Proteus spp. and other references revealed high genetic diversity for strains isolated from tree shrews, and host specificity of Proteus spp. bacteria was not found. Overall, this study provided important information on characteristics of genome for Proteus spp. isolated from tree shrews.

Genomic and functional characterization of fecal sample strains of Proteus cibarius carrying two floR antibiotic resistance genes and a multiresistance plasmid-encoded cfr gene.

The objective of this study was to investigate the molecular characteristics and horizontal transfer of florfenicol resistance gene-related sequences in Proteus strains isolated from animals. A total of six Proteus strains isolated from three farms between 2015 and 2016 were screened by polymerase chain reaction (PCR) for known florfenicol resistance genes. Proteus cibarius G11, isolated from the fecal material of a goose, was found to harbor both cfr and floR genes. Whole genome sequencing revealed that the strain harbored two copies of the floR gene: one was located on the chromosome and the other was located on a plasmid named pG11-152. Two floR-containing fragments 4028 bp in length were identical and showed transposon-like structures. The cfr gene was found on a plasmid named pG11-51 and flanked by a pair of IS26s. Thus, mobile genetic elements played an important role in floR replication and horizontal resistance gene transfer. Therefore, increasing attention should be paid to monitoring the spread of resistance genes and resistance in real time.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Analysis of CRISPR/Cas system of Proteus and the factors affected the functional mechanism.

BACKGROUND: The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. OBJECTIVES: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system. METHODS: CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. RESULTS: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. CONCLUSIONS: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.

Relationship between microbial diversity and nitrogenase activity of Stipagrostis pennata rhizosheath.

Nitrogen is the key factor for plant survival and growth, especially in the desert. Stipagrostis pennata, a sand born drought-resistant plant, could colonize pioneerly in Gurbantunggut Desert during revegetation. One strategy for their environment adaptation was the rhizosheath formatted by root-hair, mucilaginous exudates, microbial components, and soil particles, for which not only provides a favorable living microenvironment but also supplies essential nutrients. To understand the relationship between microorganisms living in rhizosheaths and the nitrogen nutrition supply, the microbial diversity and nitrogenase activity was estimated during the growth of S. pennata. Five samples of the rhizosheath, which based on the development periods of the plant, regreen, flowering, filling, seed maturating, and withering period, were collected. The nitrogenase activity was estimated by acetylene reduction and the microbial diversity was analyzed by 16S rRNA high-throughput sequencing. The results showed that the nitrogenase activity was increased slowly during regreen to flowering, while reached a peak rapidly at filling sample and then decreased gradually. A total of 274 operational taxonomic units (OTUs) were identified and significant differences in community structure and composition at each growth period. Among them, the main phyla included Actinobacteria and Proteus, which were the most abundant phyla in all periods. In addition, the microbial diversity in the grain filling period was higher than other periods in view of the analysis of alpha diversity and beta diversity. Furthermore, principal component analysis (PCA) analysis showed that the microbial communities in the filling period was low in similarity with other periods. Most importantly, the OTUs associated with nitrogen fixation is the most during the filling period, involving Phagecidae and Fucoraceae. Overall, the study not only revealed the differences in nitrogenase activity among different developmental periods in S. pennata, but also explored the potential bridges between it and community structure and diversity of bacteria.

Engineering of L-amino acid deaminases for the production of α-keto acids from L-amino acids.

α-keto acids are organic compounds that contain an acid group and a ketone group. L-amino acid deaminases are enzymes that catalyze the oxidative deamination of amino acids for the formation of their corresponding α-keto acids and ammonia. α-keto acids are synthesized industrially via chemical processes that are costly and use harsh chemicals. The use of the directed evolution technique, followed by the screening and selection of desirable variants, to evolve enzymes has proven to be an effective way to engineer enzymes with improved performance. This review presents recent studies in which the directed evolution technique was used to evolve enzymes, with an emphasis on L-amino acid deaminases for the whole-cell biocatalysts production of α-keto acids from their corresponding L-amino acids. We discuss and highlight recent cases where the engineered L-amino acid deaminases resulted in an improved production yield of phenylpyruvic acid, α-ketoisocaproate, α-ketoisovaleric acid, α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and pyruvate.

Proteae: a reservoir of class 2 integrons?

OBJECTIVES: Our aim was to confirm with a large panel of clinical isolates that class 2 integrons are highly prevalent in Proteae and to analyse their genetic characteristics. METHODS: Proteae (Proteus spp., Morganella spp. and Providencia spp.) isolates were collected from clinical samples during 2013 at Limoges University Hospital, France. The presence of class 1, 2 and 3 integrons was investigated by quantitative PCR. The presence of a stop codon in the intI2 gene was determined by Sanger sequencing. The gene cassette arrays of class 2 integrons were determined by PCR-RFLP and Sanger sequencing or next-generation sequencing when needed. RESULTS: Of the 327 Proteae collected, 103 (31.5%) harboured a class 2 integron and 45 (13.8%) a class 1 integron. No class 3 integrons were detected. One functional IntI2 integrase was detected in a Morganella morganii isolate. Six different gene cassette arrays were detected. Four had already been described in the literature: dfrA1-sat2-aadA1 (72 isolates), dfrA1-catB2-sat2-aadA1 (17), sat2-aadA1 (6) and lnu(F), dfrA1, aadA1 (1). We identified two new gene cassette arrays: (i) a new variant of the dfrA1 gene cassette (one isolate; the one with the functional IntI2); and (ii) the array dfrA1-gcu115-sat2 harbouring the new gcu115 gene cassette with two ORFs encoding proteins of unknown functions (five isolates). CONCLUSIONS: We showed a high frequency of class 2 integrons, as well as a diversity of gene cassette arrays, among Proteae. This work highlights that the Proteae tribe plays an important role as a reservoir of class 2 integrons.