Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Phylogeny of North Americas largest cicada radiation redefines Tibicinoides and Okanagana (Hemiptera: Auchenorrhyncha: Cicadidae: Tibicininae).

Tibicinoides, with three small endemic California cicada species, has a confusing, intertwined systematic history with Okanagana that we unravel here. An ingroup including all species of Tibicinoides and the majority (84.7%) of Okanagana species were sampled for six gene regions, polarized with Clidophleps, Okanagodes, Subpsaltria, and Tibicina outgroups, and subjected to Bayesian phylogenetic analysis. Although the ingroup was monophyletic from all outgroups including Tibicina, Tibicinoides rendered Okanagana paraphyletic among two major ingroup clades. To bring classification into agreement with phylogeny, we redescribe and redefine Tibicinoides to include all Okanagana species with a hooked uncus in the male genitalia, all of which grouped with the type T. cupreosparsa (Uhler, 1889) in the first of these clades: T. boweni (Chatfield-Taylor & Cole, 2020) comb. n., T. catalina (Davis, 1936) comb. n., T. hesperia (Uhler, 1876) comb. n., T. mercedita (Davis, 1915), T. minuta (Davis, 1915), T. pallidula (Davis, 1917a) comb. n., T. pernix (Bliven, 1964) comb. n., T. rubrovenosa (Davis, 1915) comb. n., T. simulata (Davis, 1921) comb. n., T. striatipes (Haldeman, 1852) comb. n., T. uncinata (Van Duzee, 1915) comb. n., T. utahensis (Davis, 1919) comb. n., and T. vanduzeei (Distant, 1914) comb. n. Okanagana is redescribed and restricted to the species of the second major clade which contained the type O. rimosa (Say, 1830). We describe two new genera for morphologically distinct orphan lineages: Chlorocanta gen. nov. for C. viridis (Davis, 1918) comb. n. and Hewlettia gen. nov. for H. nigriviridis (Davis, 1921) comb. n. We recognize O. rubrobasalis Davis, 1926 stat. rev. as a species and relegate two former species to junior subjective synonyms: O. noveboracensis (Emmons, 1854) = O. canadensis (Provancher, 1889) and O. occidentalis (Walker in Lord, 1866) = O. lurida Davis, 1919. Tibicinoides and Okanagana together represent a rapid radiation that presents challenges to phylogenetic analysis including suboptimal outgroups and short internodes.

LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron-sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 µM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

A revision of the shield-back katydid genus Neduba (Orthoptera: Tettigoniidae: Tettigoniinae: Nedubini).

The Nearctic shield-back katydid genus Neduba is revised. Species boundaries were demarcated by molecular phylogenetic analysis, morphology, quantitative analysis of calling songs, and karyotypes. Nine previously described species are redescribed: N. carinata, N. castanea, N. convexa, N. diabolica, N. extincta, N. macneilli, N. propsti, N. sierranus, and N. steindachneri, and twelve new species are described: N. ambagiosa sp. n., N. arborea sp. n., N. cascadia sp. n., N. duplocantans sp. n., N. inversa sp. n., N. longiplutea sp. n., N. lucubrata sp. n., N. oblongata sp. n., N. prorocantans sp. n., N. radicata sp. n., N. radocantans sp. n., and N. sequoia sp. n. We chose a lectotype for N. steindachneri and transferred N. picturata from a junior synonym of N. diabolica to a junior synonym of N. steindachneri. Diversification in this relict group reflects cycles of allopatric isolation and secondary contact amidst the tumultuous, evolving geography of western North America. The taxonomy and phylogenies presented in this revision lay the groundwork for studies of speciation, biogeography, hybrid zones, and behavioral evolution. Given that one Neduba species is already extinct from human environmental disturbance, we suggest conservation priorities for the genus.

Thirteen new species of Chilecicada Sanborn, 2014 (Hemiptera: Auchenorrhyncha: Cicadidae: Tibicininae) expand the highly endemic cicada fauna of Chile.

The genus Chilecicada Sanborn, 2014 is shown to be a complex of closely related species rather than a monospecific genus. Chilecicada citatatemporaria Sanborn Cole n. sp., C. culenesensis Sanborn Cole n. sp., C. curacaviensis Sanborn Cole n. sp., C. impartemporaria Sanborn Cole n. sp., C. magna Sanborn Cole n. sp., C. mapuchensis Sanborn n. sp., C. oraria Sanborn Cole n. sp., C. parrajaraorum Sanborn n. sp., C. partemporaria Sanborn Cole n. sp., C. pehuenchesensis Sanborn Cole n. sp., C. trifascia Sanborn n. sp., C. trifasciunca Sanborn Cole n. sp., and C. viridicitata Sanborn Cole n. sp. are described as new. Chilecicada occidentis Walker, 1850 is re-described to facilitate separation of the new species from the only previously known species. Song and cytochrome oxidase I analysis available for most species support the separation of the new taxa from the type species of the genus. Known species distributions and a key to the species of the genus are also provided. The new species increases the known cicada diversity 61.9% to 34 species, 91.2% of which are endemic to Chile.

A new species of Okanagana from the Walker Lane region of Nevada and California (Hemiptera: Auchenorrhyncha: Cicadidae).

Okanagana boweni sp. n. is described from the western margin of the Great Basin of North America. The new species is diagnosed from allopatric O. simulata Davis and sympatric O. utahensis Davis using morphological, bioacoustical, and molecular characters. The distribution of this new species coincides with the Walker Lane region that lies along the border of California and Nevada, USA. Based on geography, bioacoustics, morphology, and molecular phylogenetics, we hypothesize that O. boweni sp. n. is the allopatric sister species of O. simulata.

Multilocus phylogeny of Gryllus field crickets (Orthoptera: Gryllidae: Gryllinae) utilizing anchored hybrid enrichment.

We present the first comprehensive molecular phylogeny of Gryllus field cricket species found in the United States and Canada, select additional named Gryllus species found in Mexico and the Bahamas, plus the European field cricket G. campestris Linnaeus and the Afro-Eurasian cricket G. bimaculatus De Geer. Acheta, Teleogryllus, and Nigrogryllus were used as outgroups. Anchored hybrid enrichment was used to generate 492,531 base pairs of DNA sequence from 563 loci. RAxML analysis of concatenated sequence data and Astral analysis of gene trees gave broadly congruent results, especially for older branches and overall tree structure. The North American Gryllus are monophyletic with respect to the two Old World taxa; certain sub-groups show rapid recent divergence. This is the first Anchored Hybrid Enrichment study of an insect group done for closely related species within a single genus, and the results illustrate the challenges of reconstructing the evolutionary history of young rapidly diverged taxa when both incomplete lineage sorting and probable hybridization are at play. Because Gryllus field crickets have been used extensively as a model system in evolutionary ecology, behavior, neuro-physiology, speciation, and life-history and life-cycle evolution, these results will help inform, interpret, and guide future research in these areas.

How Bioinformatic Tools Guide Experiments To Resolve the Chaos of Apparently Unlimited Metabolic Variation.

Hexuronic acids, oxidation products of common sugars, are widespread in eukaryotic cells. Galacturonic acid is the main carbohydrate component of pectin found in plant cell walls and glucuronic acid is a component of proteoglycans in animals. However, despite their importance as carbohydrate substrates, metabolism of hexuronic acids has long remained a poorly studied corner of the bacterial metabolic map. In the current issue of Journal of Bacteriology, Bouvier and coworkers present a detailed analysis of genes involved in hexuronate utilization in various proteobacteria and report the verification of their bioinformatics predictions by carefully designed experiments (J. T. Bouvier et al., J Bacteriol 201:e00431-18, 2019, https://doi.org/10.1128/JB.00431-18). This study provides a solid basis for understanding hexuronate metabolism and its regulation in other bacterial phyla.

Development of a generic ß-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli.

Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 ß-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. ß-lactamase was fused to the C-terminal of scFv and ß-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for ß-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.

Anaerobic Bacterial Response to Nitrosative Stress.

This chapter provides an overview of current knowledge of how anaerobic bacteria protect themselves against nitrosative stress. Nitric oxide (NO) is the primary source of this stress. Aerobically its removal is an oxidative process, whereas reduction is required anaerobically. Mechanisms required to protect aerobic and anaerobic bacteria are therefore different. Several themes recur in the review. First, how gene expression is regulated often provides clues to the physiological function of the gene products. Second, the physiological significance of reports based upon experiments under extreme conditions that bacteria do not encounter in their natural environment requires reassessment. Third, responses to the primary source of stress need to be distinguished from secondary consequences of chemical damage due to failure of repair mechanisms to cope with extreme conditions. NO is generated by many mechanisms, some of which remain undefined. An example is the recent demonstration that the hybrid cluster protein combines with YtfE (or RIC protein, for repair of iron centres damaged by nitrosative stress) in a new pathway to repair key iron-sulphur proteins damaged by nitrosative stress. The functions of many genes expressed in response to nitrosative stress remain either controversial or are completely unknown. The concentration of NO that accumulates in the bacterial cytoplasm is essentially unknown, so dogmatic statements cannot be made that damage to transcription factors (Fur, FNR, SoxRS, MelR, OxyR) occurs naturally as part of a physiologically relevant signalling mechanism. Such doubts can be resolved by simple experiments to meet six proposed criteria.

The Essential Genome of Escherichia coli K-12.

Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCE Incentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.

Optimizing host cell physiology and stress avoidance for the production of recombinant human tumour necrosis factor α in Escherichia coli.

As high-level recombinant protein production (RPP) exerts a massive stress on the production host, an extensive literature on RPP optimization focuses on separating the growth phase from RPP production once sufficient biomass has been obtained. The aim of the current investigation was to optimize the benefits of the relatively neglected alternative strategy to achieve high-level RPP during growth by minimizing stress on the host. High yields of the biopharmaceutical recombinant human tumour necrosis factor alpha (rhTNFα) were obtained by fed-batch fermentation relevant to industrial production based upon parameters that most severely affected RPP in preliminary laboratory scale batch cultures. Decreasing the inducer concentration and growth temperature, but increasing the production period, were far more effective for increasing RPP yields than changing the growth phase at which production was induced. High yields of up to 5 g l-1 of rhTNFα were obtained with minimal plasmid loss, even in synthetic media that lack animal-derived components and are therefore fully compliant with regulatory requirements. Most of the product was soluble and biologically active. In summary, stress minimization was shown to be an effective way to optimize the production of rhTNFα. Data generated in shake-flask experiments allowed the design of intensified bioreactor cultures in which RPP and growth could be balanced, leading to higher yield of both rhTNFα and biomass than with previous fermentations. An additional benefit of this approach is avoidance of lysis during harvesting and downstream processing.

Coordinated response of the Desulfovibrio desulfuricans 27774 transcriptome to nitrate, nitrite and nitric oxide.

The sulfate reducing bacterium Desulfovibrio desulfuricans inhabits both the human gut and external environments. It can reduce nitrate and nitrite as alternative electron acceptors to sulfate to support growth. Like other sulphate reducing bacteria, it can also protect itself against nitrosative stress caused by NO generated when nitrite accumulates. By combining in vitro experiments with bioinformatic and RNA-seq data, metabolic responses to nitrate or NO and how nitrate and nitrite reduction are coordinated with the response to nitrosative stress were revealed. Although nitrate and nitrite reduction are tightly regulated in response to substrate availability, the global responses to nitrate or NO were largely regulated independently. Multiple NADH dehydrogenases, transcription factors of unknown function and genes for iron uptake were differentially expressed in response to electron acceptor availability or nitrosative stress. Amongst many fascinating problems for future research, the data revealed a YtfE orthologue, Ddes_1165, that is implicated in the repair of nitrosative damage. The combined data suggest that three transcription factors coordinate this regulation in which NrfS-NrfR coordinates nitrate and nitrite reduction to minimize toxicity due to nitrite accumulation, HcpR1 serves a global role in regulating the response to nitrate, and HcpR2 regulates the response to nitrosative stress.

Living rain gauges: cumulative precipitation explains the emergence schedules of California protoperiodical cicadas.

Mass multi-species cicada emergences (broods) occur in California with variable periodicity. Here we present the first rule set that predicts the emergence of protoperiodical cicada communities. We tested two hypotheses with a dataset consisting of direct observations and georeferenced museum specimen records: first, that cicada broods are triggered to emerge by periodic ENSO events and second, that brood emergences occur after precipitation accumulates above a threshold value. The period of ENSO events does not explain the observed pattern of cicada brood emergence. Rather, broods emerged given two conditions: (1) that total precipitation exceeded a threshold of 1,181 mm, and (2) that a minimum 3-yr period lapsed. The precipitation threshold is obeyed over an 800 km north-south distance in California and across a variety of habitats. We predict the next brood emergence at one study site in arid Los Angeles County desert foothills to occur in 2020 or, if drought conditions continue, in 2021.

MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis.

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain.

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5 144 392 bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2 803 932 bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577 351 bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157 : H7; therefore, it is not an ancestral state for the species.

A new species of Megatibicen endemic to Mescalero-Monahans shinnery sands (Hemiptera: Auchenorrhyncha: Cicadidae).

Megatibicen harenosus sp. n. is described from the Mescalero-Monahans shinnery sands of New Mexico and Texas, U.S.A. The new species is diagnosed from similar species, especially M. tremulus which it resembles closely, by male genital morphology, color pattern, calling song, and ecology. Seven characters from the male calling song are described from analysis of field recordings, of which all four temporal song characters are significantly different from M. tremulus. With one of the most southwestern distribution of any Megatibicen species, M. harenosus is a new addition to the rich, endemic, and understudied Mescalero-Monahans shinnery sands biota. The possibility that M. harenosus and M. tremulus are sister species is raised. The ecological, biological, and evolutionary species concepts support species status for M. harenosus, and an hypothesis of peripatric speciation in peripheral isolation is advanced.

Regulation, sensory domains and roles of two Desulfovibrio desulfuricans ATCC27774 Crp family transcription factors, HcpR1 and HcpR2, in response to nitrosative stress.

In silico analyses identified a Crp/Fnr family transcription factor (HcpR) in sulfate-reducing bacteria that controls expression of the hcp gene, which encodes the hybrid cluster protein and contributes to nitrosative stress responses. There is only one hcpR gene in the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, but two copies in Desulfovibrio desulfuricans 27774, which can use nitrate as an alternative electron acceptor to sulfate. Structures of the D. desulfuricans hcpR1, hcpR2 and hcp operons are reported. We present evidence that hcp expression is regulated by HcpR2, not by HcpR1, and that these two regulators differ in both their DNA-binding site specificity and their sensory domains. HcpR1 is predicted to be a b-type cytochrome. HcpR1 binds upstream of the hcpR1 operon and its synthesis is regulated coordinately with hcp in response to NO. In contrast, hcpR2 expression was not induced by nitrate, nitrite or NO. HcpR2 is an iron-sulfur protein that reacts with NO and O2 . We propose that HcpR1 and HcpR2 use different sensory mechanisms to regulate subsets of genes required for defense against NO-induced nitrosative stress, and that diversification of signal perception and DNA recognition by these two proteins is a product of D. desulfuricans adaptation to its particular environmental niche.