RNA-seq reveals multifaceted gene expression response to Fab production in Escherichia coli fed-batch processes with particular focus on ribosome stalling.

BACKGROUND: Escherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations. RESULTS: Production of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg2+, indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising. CONCLUSIONS: Through characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production.

Recommendations for psychological assessment before bariatric surgery.

This document was created based on the need to standardize the psychological examination procedure prior to bariatric surgery. A valuable inspiration was the recommendations issued by the American Society for Metabolic and Bariatric Surgery. Bariatric or metabolic surgery has an undeniable positive effect in the treatment of obesity, in terms of improving somatic diseases, psychological disorders and psychosocial functioning. At the same time, it introduces major changes in the individual's life to which he or she must adapt. The treatment of obesity by surgery requires a fundamental change in lifestyle and the lifelong cooperation of the patient with the entire therapeutic team. Psychological care is a standard part of the entire treatment process. The role of the psychologist is not exclusively diagnostic. In indicated cases, it offers preoperative and postoperative psychological intervention, education and cooperation in the development of an individual treatment plan. Its aim is to deepen the patient's motivation to comply with dietary and regimen recommendations and to provide psychological support in the event of worsening psychological difficulties.

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield.

Antibody fragments such as Fab's require the formation of disulfide bonds to achieve a proper folding state. During their recombinant, periplasmic expression in Escherichia coli, oxidative folding is mediated by the DsbA/DsbB system in concert with ubiquinone. Thereby, overexpression of Fab's is linked to the respiratory chain, which is not only immensely important for the cell's energy household but also known as a major source of reactive oxygen species. However, the effects of an increased oxidative folding demand and the consequently required electron flux via ubiquinone on the host cell have not been characterized so far. Here, we show that Fab expression in E. coli BL21(DE3) interfered with the intracellular redox balance, thereby negatively impacting host cell performance. Production of four different model Fab's in lab-scale fed-batch cultivations led to increased oxygen consumption rates and strong cell lysis. An RNA sequencing analysis revealed transcription activation of the oxidative stress-responsive soxS gene in the Fab-producing strains. We attributed this to the accumulation of intracellular superoxide, which was measured using flow cytometry. An exogenously supplemented ubiquinone analogue improved Fab yields up to 82%, indicating that partitioning of the quinone pool between aerobic respiration and oxidative folding limited ubiquinone availability and hence disulfide bond formation capacity. Combined, our results provide a more in-depth understanding of the profound effects that periplasmic Fab expression and in particular disulfide bond formation has on the host cell. Thereby, we show new possibilities to elaborate cell engineering and process strategies for improved host cell fitness and process outcome.

Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing.

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed-batch-like microbioreactor cultivations are conducted under production conditions. The easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole-genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21Q expression system remains stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives have positive effects on long-term stability. The results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial-relevant production conditions is an efficient strain development tool for production hosts.

Adult obese patient in primary care.

Overweight and obesity prevalence in middle aged subjects in the Czech Republic is more than 50 per cent, obesity is found in around 26 per cent of population. Obesity management is a long-term and time-consuming process. Early start of the treatment can prevent continuous weight gain and development of co-morbidities. General practitioners see obese patients usually as the first and they represent the first point of contact for adults with obesity. The basis of obesity management is a change of the lifestyle with added pharmacotherapy and/or bariatric/metabolic surgery. The paper presents overview of methods in obesity diagnostics and management and possibilities of their use in GPs daily practice.

Psychological aspects of obesity.

Obesity is a metabolic disorder conditioned by several factors with the individual genetic proneness to accumulation of body fat with a positive energetic balance. If such definition describes the essential nature of obesity aptly, the treatment thereof ought to be the realm of somatic medicine and somatically oriented physicians, which is, unfortunately, frequently the case. Yet, not only being a disorder concerning improper body composition, but also a difference in cognitive processes and emotions of the obese, obesity needs to be considered in a more complex manner. The life of the obese consists of periods of strict, starvation diets on one hand and total loss of control and excessive calorie intake. Therefore, the corresponding therapy also needs to be provided in a more complex fashion, i.e. it is not solely the somatic disorder that should be addressed, but also the emotions and cognitions which induce the undesirable behaviour. Generally, it is possible to summarise that cognition and emotions are likely to be anticipated, directed and controlled by affecting the stimuli promoting the erratic attitude. Thanks to the achievements which relate not only to loss of weight, but also to higher self-esteem, more gratifying feelings aroused by the patients self, improvement of both physical and mental conditions and enhancement of the quality of life as a whole, the new behaviour patterns are established, strengthened and sustained on a long-term basis. Several psychotherapeutic attitudes/methods may be used with cognitive-behavioral therapy, existential therapy and, recently, psychodynamic approach. The psychologists role is essential and fundamental in both conservative and the metabolic-bariatric treatment of obesity. The most common character traits of obese patients include predominantly neuroticism, which comprises anxiety, depressions, impulsiveness, anger and hostility. Likewise, obesity is often suffered from by children and adolescents and its treatment relies on, in like manner as with adults, an active change of unsuitable dietary and movement habits with the family of the patient and their motivation of the patient to make the desirable change. It needs to be noted, however, that except for the changes in lifestyle, treatment of psychological difficulties accompanying obesity is a part of psychotherapy of such a group of young and adolescent patients.

Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems.

BACKGROUND: The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. RESULTS: We showed that, in genome-integrated E. coli expression systems that used σ70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. CONCLUSIONS: Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Complete Genome Sequences of pLMA1 and pLMA7, Two Large Linear Plasmids of Micrococcus Strains Isolated from a High-Altitude Lake in Argentina.

The two linear plasmids pLMA1 (109,112 bp) and pLMA7 (82,075 bp) from Micrococcus strains were isolated from a high-altitude lake in the Argentinean Puna, sequenced, and annotated. These extrachromosomal elements are probably conjugative and harbor genes potentially involved in coping with the harsh conditions in such extreme environments.

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii.

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction.

Strategies and approaches in plasmidome studies-uncovering plasmid diversity disregarding of linear elements?

The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which-despite their frequent occurrence in a large number of bacteria-are largely neglected in prevalent plasmidome conceptions.

Immuno-affinity purification of PglPGIP1, a polygalacturonase-inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen.

Polygalacturonase-inhibitor proteins (PGIPs) are important plant defense proteins which modulate the activity of microbial polygalacturonases (PGs) leading to elicitor accumulation. Very few studies have been carried out towards understanding the role of PGIPs in monocot host defense. Hence, present study was taken up to characterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native glycosylated PGIP (PglPGIP1) of ~43 kDa and pI 5.9 was immunopurified from pearl millet. Comparative inhibition studies involving PglPGIP1 and its non-glycosylated form (rPglPGIP1; recombinant pearl millet PGIP produced in Escherichia coli) against two PGs, PG-II isoform from Aspergillus niger (AnPGII) and PG-III isoform from Fusarium moniliforme, showed both PGIPs to inhibit only AnPGII. The protein glycosylation was found to impact only the pH and temperature stability of PGIP, with the native form showing relatively higher stability to pH and temperature changes. Temporal accumulation of both PglPGIP1 protein (western blot and ELISA) and transcripts (real time PCR) in resistant and susceptible pearl millet cultivars showed significant Sclerospora graminicola-induced accumulation only in the incompatible interaction. Further, confocal PGIP immunolocalization results showed a very intense immuno-decoration with highest fluorescent intensities observed at the outer epidermal layer and vascular bundles in resistant cultivar only. This is the first native PGIP isolated from millets and the results indicate a role for PglPGIP1 in host defense. This could further be exploited in devising pearl millet cultivars with better pathogen resistance.

Mode of action of Bacillus licheniformis pectin methylesterase on highly methylesterified and acetylated pectins.

A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme (BliPME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50°C. BliPME is able to release up to 100% of the methylesters from lime pectin (DM 34-76→DM 0) and up to 73% of all methylesters from SBPs (DM 30-73→DM 14). BliPME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the BliPME-modified pectins using HILIC-MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of BliPME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME.

Characterization of an acetyl esterase from Myceliophthora thermophila C1 able to deacetylate xanthan.

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC-HILIC-ELSD/ESI-MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.

Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases.

Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the plant cell wall by degrading homopolygalacturonan, a major constituent of pectin. Plants fight back by employing polygalacturonase-inhibitor proteins (PGIPs). The present study compared the inhibition potential of pearl millet PGIP (Pennisetum glaucum; PglPGIP1) with the known inhibition of Phaseolus vulgaris PGIP (PvPGIP2) against two PGs, the PG-II isoform from Aspergillus niger (AnPGII) and the PG-III isoform from Fusarium moniliforme (FmPGIII). The key rationale was to elucidate the relationship between the extent of sequence similarity of the PGIPs and the corresponding PG inhibition potential. First, a pearl millet pgip gene (Pglpgip1) was isolated and phylogenetically placed among monocot PGIPs alongside foxtail millet (Setaria italica). Upstream sequence analysis of Pglpgip1 identified important cis-elements responsive to light, plant stress hormones, and anoxic stress. PglPGIP1, heterologously produced in Escherichia coli, partially inhibited AnPGII non-competitively with a pH optimum between 4.0 and 4.5, and showed no inhibition against FmPGIII. Docking analysis showed that the concave surface of PglPGIP1 interacted strongly with the N-terminal region of AnPGII away from the active site, whereas it weakly interacted with the C-terminus of FmPGIII. Interestingly, PglPGIP1 and PvPGIP2 employed similar motif regions with few identical amino acids for interaction with AnPGII at non-substrate-binding sites; however, they engaged different regions of AnPGII. Computational mutagenesis predicted D126 (PglPGIP1)-K39 (AnPGII) to be the most significant binding contact in the PglPGIP1-AnPGII complex. Such protein-protein interaction studies are crucial in the future generation of designer host proteins for improved resistance against ever-evolving pathogen virulence factors.

Extrachromosomal genetic elements in Micrococcus.

Micrococci are Gram-positive G + C-rich, nonmotile, nonspore-forming actinomycetous bacteria. Micrococcus comprises ten members, with Micrococcus luteus being the type species. Representatives of the genus play important roles in the biodegradation of xenobiotics, bioremediation processes, production of biotechnologically important enzymes or bioactive compounds, as test strains in biological assays for lysozyme and antibiotics, and as infective agents in immunocompromised humans. The first description of plasmids dates back approximately 28 years, when several extrachromosomal elements ranging in size from 1.5 to 30.2 kb were found in Micrococcus luteus. Up to the present, a number of circular plasmids conferring antibiotic resistance, the ability to degrade aromatic compounds, and osmotolerance are known, as well as cryptic elements with unidentified functions. Here, we review the Micrococcus extrachromosomal traits reported thus far including phages and the only quite recently described large linear extrachromosomal genetic elements, termed linear plasmids, which range in size from 75 kb (pJD12) to 110 kb (pLMA1) and which confer putative advantageous capabilities, such as antibiotic or heavy metal resistances (inferred from sequence analyses and curing experiments). The role of the extrachromosomal elements for the frequently proven ecological and biotechnological versatility of the genus will be addressed as well as their potential for the development and use as genetic tools.

Copy number determination, expression analysis of genes potentially involved in replication, and stability assays of pAL1--the linear megaplasmid of Arthrobacter nitroguajacolicus Rü61a.

pAL1 of the actinomycete Arthrobacter nitroguajacolicus Rü61a, a 113-kb catabolic linear megaplasmid of the invertron-type, enables its host to utilize quinaldine (or 4-hydroxyquinaldine) as the sole carbon and energy source. Comparative Southern hybridization and quantitative real-time PCR analyses revealed copy numbers of two for cells grown in minimal medium supplemented with 4-hydroxyquinaldine and four for cells cultivated in Luria-Bertani medium. However, medium-dependent differences in the expression levels of genes potentially involved in plasmid replication were not seen. As replication and maintenance of invertrons may depend on single-stranded DNA-binding proteins (SSBs), ORF39, the gene product of which displaying similarities to SSBs, was knocked out. However, structural stability and replication of pAL1 was not influenced. When plasmid maintenance of LB-grown wild-type cells was assessed, it was seen that after approx. 200 generations almost 80% of the cells were devoid of plasmid, indicating segregational instability of pAL1.

Replication-involved genes of pAL1, the linear plasmid of Arthrobacter nitroguajacolicus Rü61a--phylogenetic and transcriptional analysis.

The 113-kb pAL1 is the only Arthrobacter linear plasmid known; it has terminal inverted repeats and 5' covalently attached terminal proteins (TPs). The latter and a telomere-associated protein (Tap) are encoded by plasmid ORFs 102 and 101, respectively. As for Streptomyces linear replicons, in which both above proteins are instrumental in telomere patching, they are involved in pAL1 replication as well. However, the alignment of actinobacterial Taps and TPs revealed that pAL1 and the linear elements from Rhodococci comprise a discrete phylogenetic group, clearly delineated from the streptomycetes linear plasmids. In line with such findings is the same genetic arrangement of ORF 101 and 102 counterparts in the rhodococcal elements. Furthermore, the adjacent gene (ORF100) has matches in the rhodococcal plasmids as well. In linear elements of Streptomyces there is no ORF100 homolog. Two alternative annotations are possible for ORF100 gene products. As RT-PCR revealed cotranscription of ORFs 100-102, the ORF100 gene product is presumably involved in replicative processes. Taken also into consideration the likely absence of an internal replication origin (other than in Streptomyces linear elements), we assume a distinct replication/telomere patching mechanism for pAL1 type replicons.

Structural peculiarities of linear megaplasmid, pLMA1, from Micrococcus luteus interfere with pyrosequencing reads assembly.

Different strains of Micrococcus luteus, isolated from high-altitude Argentinean wetlands, were recently reported to harbour the linear plasmids pLMA1, pLMH5 and pLMV7, all of which with 5'-covalently attached terminal proteins. The link between pLMA1 and the host's erythromycin resistance as well as further presumptive qualities prompted us to perform a detailed characterization. When the 454 technology was applied for direct sequencing of gel-purified pLMA1, assembly of the reads was impossible. However, combined Sanger/454 sequencing of cloned pLMA1 fragments, covering altogether 23 kb of the 110-kb spanning plasmid, allowed numerous sequence repeats of varying in lengths to be identified thus rendering an explanation for the above 454 assembly failure. A large number of putative transposase genes were identified as well. Furthermore, a region with five putative iteron sequences is possibly involved in pLMA1 replication.

Novel linear megaplasmid from Brevibacterium sp. isolated from extreme environment.

Brevibacterium sp. Ap13, isolated from flamingo's feces in Laguna Aparejos, a high-altitude lake located at approximately 4,200 m in the northwest of Argentina was previously found to be resistant to multiple antibiotics, and was therefore screened for plasmids that may be implicated in antibiotic resistance. Brevibacterium sp. Ap13 was found to contain two plasmids of approximately 87 and 436 kb, designated pAP13 and pAP13c, respectively. Only pAP13 was stably maintained and was extensively characterized by pulsed-field gel electrophoresis to reveal that this plasmid is linear and likely has covalently linked terminal proteins associated with its 5' ends. This is the first report of a linear plasmid in the genus Brevibacterium and may provide a new tool for genetic manipulation of this commercially important genus.

First report of linear megaplasmids in the genus Micrococcus.

High-altitude wetlands (above 4200m) in the northwest of Argentina are considered pristine and extreme environments. Micrococcus sp. A1, H5, and V7, isolated from such environments, were shown to contain linear megaplasmids, designated pLMA1, pLMH5, and pLMV7, respectively. As known from linear plasmids of other actinomycetes, all three plasmids were resistant to lambda exonuclease treatment, which is consistent with having terminal proteins covalently attached to their 5' DNA ends. Electrophoretic mobility, Southern analysis, and restriction endonuclease patterns revealed pLMA1 and pLMH5 being indistinguishable plasmids, even though they were found in different strains isolated from two distant wetlands - Laguna Azul and Laguna Huaca Huasi. Analysis of 16S rDNA sequences of Micrococcus sp. A1, H5, and V7 suggested a close relationship to Micrococcus luteus. Typing of isolates was performed using fingerprint patterns generated by BOX-PCR. Plasmid-deficient strains, generated from Micrococcus sp. A1, showed a significantly decreased resistance level for erythromycin.