Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110

Schaffert L, Schneiker-Bekel S, Gierhake J, et al. Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110. Applied Microbiology and Biotechnology. 2020;104:5395–5408.Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8–16% higher product titers when grown in maltose-containing medium

Complete genome sequence and lifestyle of black-pigmented Corynebacterium aurimucosum ATCC 700975 (formerly C. nigricans CN-1) isolated from a vaginal swab of a woman with spontaneous abortion

Trost E, Götker S, Schneider J, et al. Complete genome sequence and lifestyle of black-pigmented Corynebacterium aurimucosum ATCC 700975 (formerly C. nigricans CN-1) isolated from a vaginal swab of a woman with spontaneous abortion. BMC Genomics. 2010;11(1): 91.Background Corynebacterium aurimucosum is a slightly yellowish, non-lipophilic, facultative anaerobic member of the genus Corynebacterium and predominantly isolated from human clinical specimens. Unusual black-pigmented variants of C. aurimucosum (originally named as C. nigricans) continue to be recovered from the female urogenital tract and they are associated with complications during pregnancy. C. aurimucosum ATCC 700975 (C. nigricans CN-1) was originally isolated from a vaginal swab of a 34-year-old woman who experienced a spontaneous abortion during month six of pregnancy. For a better understanding of the physiology and lifestyle of this potential urogenital pathogen, the complete genome sequence of C. aurimucosum ATCC 700975 was determined. Results Sequencing and assembly of the C. aurimucosum ATCC 700975 genome yielded a circular chromosome of 2,790,189 bp in size and the 29,037-bp plasmid pET44827. Specific gene sets associated with the central metabolism of C. aurimucosum apparently provide enhanced metabolic flexibility and adaptability in aerobic, anaerobic and low-pH environments, including gene clusters for the uptake and degradation of aromatic amines, L-histidine and L-tartrate as well as a gene region for the formation of selenocysteine and its incorporation into formate dehydrogenase. Plasmid pET44827 codes for a non-ribosomal peptide synthetase that plays the pivotal role in the synthesis of the characteristic black pigment of C. aurimucosum ATCC 700975. Conclusions The data obtained by the genome project suggest that C. aurimucosum could be both a resident of the human gut and possibly a pathogen in the female genital tract causing complications during pregnancy. Since hitherto all black-pigmented C. aurimucosum strains have been recovered from female genital source, biosynthesis of the pigment is apparently required for colonization by protecting the bacterial cells against the high hydrogen peroxide concentration in the vaginal environment. The location of the corresponding genes on plasmid pET44827 explains why black-pigmented (formerly C. nigricans) and non-pigmented C. aurimucosum strains were isolated from clinical specimens

Factores que influyen en la dispersión espacio-temporal de broca del café Hypothenemus hampei (Coleoptera: Curculionidae)

La broca del café, Hypothenemus hampei (Ferrari), es la plaga más importante del café, Coffea spp., en el mundo. Este insecto ha sido motivo de numerosos estudios, sin embargo, muchos aspectos de su dispersión se desconocen o requieren ser determinados. La presente investigación tuvo el objetivo de estudiar los factores que influyen en la dispersión espacio-temporal de la broca del café. Dado que la dispersión es un proceso complejo, para ser estudiada se desarrollaron técnicas para la obtención del material biológico con condiciones adecuadas para el estudio, para facilitar el estudio de la morfología del complejo espermático de H. hampei y de marcaje-liberación-recaptura para estudiar la capacidad de vuelo y los factores que la afectan. Los resultados más sobresalientes indican que la espermateca de H. hampei está ubicada en la parte final del abdomen entre la glándula accesoria y el oviducto común y está compuesta por un ducto espermático, músculos espermáticos y una glándula espermática; además, posee músculos al final de su curvatura extrema y su cutícula está finamente reticulada. Los espermatozoides se observaron como filamentos minúsculos, endebles y homogéneos, congregados longitudinalmente dentro de la espermateca. La emergencia masiva de las CH se relacionó con las lluvias y se presentó entre febrero y junio, que corresponde al periodo intercosecha. Todas las CH examinadas tenían espermatozoides en la espermateca, lo q ue sugiere que se habían apareado antes de abandonar el fruto natal. La sobrevivencia de las hembras colonizadoras fue mayor en ambientes humedos y en sustratos diferentes a su hospedero. Durante el periodo de fructificación del café, las hembras se dispersaron mediante vuelo a los glomérulos de frutos adyacentes. Después de colonizar un fruto, la CH perdió su capacidad de vuelo de forma gradual en el transcurso de 5 a 6 días; durante este tiempo, las hembras evaluadas realizar más de un vuelo. Algunas CH marcadas y liberadas se recapturaron a 75 m de distancia del sitio de liberación a las 24 h después de haber sido liberadas. Finalmente, se discute la importancia de los hallazgos de la dispersión espacio-temporal de la broca del café, como información clave para mejorar la comprensión del complejo café-broca, y el manejo de la broca. (Résumé d'auteur

The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110

Wolf T, Droste J, Gren T, et al. The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110. BMC Genomics. 2017;18(1): 562.Background Acarbose is used in the treatment of diabetes mellitus type II and is produced by Actinoplanes sp. SE50/110. Although the biosynthesis of acarbose has been intensively studied, profound knowledge about transcription factors involved in acarbose biosynthesis and their binding sites has been missing until now. In contrast to acarbose biosynthetic gene clusters in Streptomyces spp., the corresponding gene cluster of Actinoplanes sp. SE50/110 lacks genes for transcriptional regulators. Results The acarbose regulator C (AcrC) was identified through an in silico approach by aligning the LacI family regulators of acarbose biosynthetic gene clusters in Streptomyces spp. with the Actinoplanes sp. SE50/110 genome. The gene for acrC, located in a head-to-head arrangement with the maltose/maltodextrin ABC transporter malEFG operon, was deleted by introducing PCR targeting for Actinoplanes sp. SE50/110. Characterization was carried out through cultivation experiments, genome-wide microarray hybridizations, and RT-qPCR as well as electrophoretic mobility shift assays for the elucidation of binding motifs. The results show that AcrC binds to the intergenic region between acbE and acbD in Actinoplanes sp. SE50/110 and acts as a transcriptional repressor on these genes. The transcriptomic profile of the wild type was reconstituted through a complementation of the deleted acrC gene. Additionally, regulatory sequence motifs for the binding of AcrC were identified in the intergenic region of acbE and acbD. It was shown that AcrC expression influences acarbose formation in the early growth phase. Interestingly, AcrC does not regulate the malEFG operon. Conclusions This study characterizes the first known transcription factor of the acarbose biosynthetic gene cluster in Actinoplanes sp. SE50/110. It therefore represents an important step for understanding the regulatory network of this organism. Based on this work, rational strain design for improving the biotechnological production of acarbose can now be implemented

Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110

Schaffert L, März C, Burkhardt L, et al. Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. Microbial Cell Factories. 2019;18(1): 114.Background Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. Results The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. Conclusion Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae

Gross R, Guzman CA, Sebaihia M, et al. The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae. BMC Genomics. 2008;9(1): 449.Background: Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. Results: In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. Conclusion: The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches

Das konjugative Hg-Resistenzplasmid pSB102 aus der bakteriellen Gemeinschaft der Luzernenrhizosphäre: Isolierung, Sequenzierung und Sequenzinterpretation

Schneiker-Bekel S. Das konjugative Hg-Resistenzplasmid pSB102 aus der bakteriellen Gemeinschaft der Luzernenrhizosphäre: Isolierung, Sequenzierung und Sequenzinterpretation. Bielefeld; 2001

The Sinorhizobium meliloti insertion sequence (IS) element ISRm14 is related to a previously unrecognized IS element located adjacent to the Escherichia coli locus of enterocyte effacement (LEE) pathogenicity island

Schneiker-Bekel S, Kosier B, Pühler A, Selbitschka W. The Sinorhizobium meliloti insertion sequence (IS) element ISRm14 is related to a previously unrecognized IS element located adjacent to the Escherichia coli locus of enterocyte effacement (LEE) pathogenicity island. CURRENT MICROBIOLOGY. 1999;39(5):274-281.ISRm14 is 2695 basepairs (bp) in size and bordered by 22 bp imperfect inverted repeats (IRs). A 9-bp target sequence is duplicated upon ISRm14 transposition. The DNA strand that putatively encodes the transposase enzyme carries three open reading frames (ORFs) designated ORFs 1 to 3, which specify putative proteins of 15.9 kDa, 13.1 kDa, and 61.1 kDa, respectively. According to its structural characteristics, ISRm14 belongs to the recently proposed IS66 family of IS elements. The ORFs1 to 3 encoded putative proteins displayed significant similarities to ORFs of the previously unrecognized IS element ISEc8, which is inserted adjacent to the locus of enterocyte effacement (LEE) pathogenicity island of Escherichia coli EDL933. Analyses of the distribution of ISRm14 in a natural S. meliloti population showed its widespread occurrence in 66% of the strains tested with a copy number ranging from 1 to 6

Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment

Stiens M, Schneiker-Bekel S, Pühler A, Schlüter A. Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment. FEMS MICROBIOLOGY LETTERS. 2007;271(2):297-309.The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid