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.

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.

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.

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.

The roles of the hybrid cluster protein, Hcp and its reductase, Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress.

The hybrid cluster protein, Hcp, contains a 4Fe-2S-2O iron-sulfur-oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N2 O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron-sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp-hcr operon encodes an NADH-dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co-purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO.

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.

Control of hexuronate metabolism in Escherichia coli by the two interdependent regulators, ExuR and UxuR: derepression by heterodimer formation.

Two homologous proteins, UxuR and ExuR, were previously predicted to repress synthesis of enzymes required for hexuronic acid metabolism, but little is known about the relative roles of these proteins in gene regulation. We confirmed the previous report that UxuR is essential for rapid growth with d-glucuronate as the primary source of carbon and energy. In contrast, an exuR mutant grew more rapidly on d-glucuronate than the parent. Transcription of exuR is initiated at a σ70-dependent promoter predicted in silico. Purified ExuR bound to the exuR regulatory region in the presence, but not in the absence, of d-glucuronate. Apparently weaker UxuR binding in the presence of glucuronate was also detected, and its addition decreased ExuR binding by forming ExuR-UxuR heterodimers. Glucuronate induced exuR transcription in the parental strain, but not in the exuR mutant. No evidence was obtained for cAMP-dependent regulation of exuR by the catabolite repressor protein (CRP). A previous study reported that the divergent yjjM and yjjN genes, essential for l-galactonate metabolism, are repressed by UxuR. We showed that ExuR binds to the yjjM-yjjN regulatory region, and that the binding is also glucuronate-dependent. As for the exuR promoter, UxuR appeared to decrease ExuR binding. ExuR is required for glucuronate induction of yjjM and yjjN, and CRP is required for their transcription. The combined data established that UxuR and ExuR fulfil contrasting roles in regulating hexuronic acid metabolism and indicate that ExuR can function as a transcription activator, possibly by inactivating the repressor function of UxuR by heterodimer formation.

A critical role for the cccA gene product, cytochrome c2, in diverting electrons from aerobic respiration to denitrification in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.

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.

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.

Legless pathogens: how bacterial physiology provides the key to understanding pathogenicity.

This review argues that knowledge of microbial physiology and metabolism is a prerequisite to understanding mechanisms of pathogenicity. The ability of Neisseria gonorrhoeae to cope with stresses such as those found during infection requires a sialyltransferase to sialylate its lipopolysaccharide using host-derived CMP-NANA in the human bloodstream, the ability to oxidize lactate that is abundant in the human body, outer-membrane lipoproteins that provide the first line of protection against oxidative and nitrosative stress, regulation of NO reduction independently from the nitrite reductase that forms NO, an extra haem group on the C-terminal extension of a cytochrome oxidase subunit, and a respiratory capacity far in excess of metabolic requirements. These properties are all normal components of neisserial physiology; they would all fail rigid definitions of a pathogenicity determinant. In anaerobic cultures of enteric bacteria, duplicate pathways for nitrate reduction to ammonia provide a selective advantage when nitrate is either abundant or scarce. Selection of these alternative pathways is in part regulated by two parallel two-component regulatory systems. NarX-NarL primarily ensures that nitrate is reduced in preference to thermodynamically less favourable terminal electron acceptors, but NarQ-NarP facilitates reduction of limited quantities of nitrate or other, less favourable, terminal electron acceptors in preference to fermentative growth. How enteric bacteria repair damage caused by nitrosative and oxidative damage inflicted by host defences is less well understood. In both N. gonorrhoeae and Escherichia coli, parallel pathways that duplicate particular biochemical functions are far from redundant, but fulfil specific physiological roles.

A generalised module for the selective extracellular accumulation of recombinant proteins.

BACKGROUND: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

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.

Nitrosative stress in Escherichia coli: reduction of nitric oxide.

The ability of enteric bacteria to protect themselves against reactive nitrogen species generated by their own metabolism, or as part of the innate immune response, is critical to their survival. One important defence mechanism is their ability to reduce NO (nitric oxide) to harmless products. The highest rates of NO reduction by Escherichia coli K-12 were detected after anaerobic growth in the presence of nitrate. Four proteins have been implicated as catalysts of NO reduction: the cytoplasmic sirohaem-containing nitrite reductase, NirB; the periplasmic cytochrome c nitrite reductase, NrfA; the flavorubredoxin NorV and its associated oxidoreductase, NorW; and the flavohaemoglobin, Hmp. Single mutants defective in any one of these proteins and even the mutant defective in all four proteins reduced NO at the same rate as the parent. Clearly, therefore, there are mechanisms of NO reduction by enteric bacteria that remain to be characterized. Far from being minor pathways, the currently unknown pathways are adequate to sustain almost optimal rates of NO reduction, and hence potentially provide significant protection against nitrosative stress.

An HcpR homologue from Desulfovibrio desulfuricans and its possible role in nitrate reduction and nitrosative stress.

The Escherichia coli CRP (cAMP receptor protein), is a global regulator of transcription that modulates gene expression by activation or repression at a range of promoters in E. coli. A major function is to regulate the selection of nutrients required for growth. The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC27774 is capable of utilizing sulfate, nitrite and nitrate as terminal electron acceptors. In the presence of both sulfate and nitrate, sulfate is reduced preferentially despite nitrate being the thermodynamically more favourable electron acceptor. Three inverted repeat sequences upstream of the D. desulfuricans ATCC27774 nap (nitrate reduction in the periplasm) operon have high levels of similarity to the consensus sequence for the E. coli CRP DNA-binding site. In other Desulfovibrio species a putative CRP homologue, HcpR [regulator of hcp (hybrid cluster protein) transcription], has a predicted regulon comprising genes involved in sulfate reduction and nitrosative stress. The presence of CRP consensus sites within the D. desulfuricans ATCC27774 nap promoter prompted a search for CRP homologues in the genomes of sulfate-reducing bacteria. This revealed the presence of a potential CRP homologue that we predict binds to CRP consensus sites such as those of the nap operon. Furthermore, we predict that much of the core HcpR regulon predicted in other Desulfovibrio species is conserved in D. desulfuricans.

Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA.

Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.

Enzymology and ecology of the nitrogen cycle.

The nitrogen cycle describes the processes through which nitrogen is converted between its various chemical forms. These transformations involve both biological and abiotic redox processes. The principal processes involved in the nitrogen cycle are nitrogen fixation, nitrification, nitrate assimilation, respiratory reduction of nitrate to ammonia, anaerobic ammonia oxidation (anammox) and denitrification. All of these are carried out by micro-organisms, including bacteria, archaea and some specialized fungi. In the present article, we provide a brief introduction to both the biochemical and ecological aspects of these processes and consider how human activity over the last 100 years has changed the historic balance of the global nitrogen cycle.

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.

Sense and nonsense from a systems biology approach to microbial recombinant protein production.

The 'Holy Grail' of recombinant protein production remains the availability of generic protocols and hosts for the production of even the most difficult target products. The present review provides first an explanation why the shock imposed on bacteria using a standard induction protocol not only arrests growth, but also decreases the number of colony-forming units by several orders of magnitude. Particular emphasis is placed on findings of numerous genome-wide transcriptomic studies that highlight cellular stress, in which the general stress, heat-shock and stringent responses are the underlying basis for the manifestation of the deterioration of cell physiology. We then review common approaches used to solve bottlenecks in protein folding and post-translational modification that result in recombinant protein deposition in cytoplasmic inclusion bodies. Finally, we suggest a generic approach to process design that minimizes stress on the production host and a strategy for isolating improved hosts.