Standard Laboratory Growth Conditions Research Articles

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach.

With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter.

ABSTRACTNucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus.IMPORTANCE Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory effect on the expression of the kdp genes encoding a potassium transporter. c-di-AMP binds to the USP domain of KdpD, thus providing for the first time evidence for the ability of such a domain to bind a cyclic dinucleotide.

Auxin Binding Protein 1 (ABP1): a matter of fact.

Science is a global community enterprise.Collectively,we contribute to building the knowledge tower by using bricks and mortar that should be strong,solid,and long lasting.A brick with defects may lead this tower,or a part there of,to collapse.When that happens,the community suffers,laying waste countless dedicated hours of work,especially by students and postdoctoral scholars.This is the reason ary

Microarray transposon tracking for the mapping of conditionally essential genes in Campylobacter jejuni.

Although whole genome approaches to the study of bacteria have grown substantially in the past decade, there is still a need for quick and easy methods for the determination of which genes are essential for the growth of these bacteria under specific growth conditions. There are numerous methods to accomplish this depending on the resources and equipment available, each with their own advantages and disadvantages. Here we describe a method we successfully employed to map the essential genes of Campylobacter jejuni using a microarray transposon tracking approach where we constructed a saturated transposon mutant library in the C. jejuni strain NCTC11168 and used a genomic microarray approach to identify genes lacking transposon insertions under standard laboratory growth conditions. With a fully saturated library, the absence of transposon insertions can be used as an indicator of a gene essential for the survival and growth for the conditions used for the mutant library.

The exo-proteome and exo-metabolome of Nostoc punctiforme (Cyanobacteria) in the presence and absence of nitrate

The ability of nitrogen-fixing filamentous Cyanobacteria to adapt to multiple environments comes in part from assessing and responding to external stimuli, an event that is initiated in the extracellular milieu. While it is known that these organisms produce numerous extracellular substances, little work has been done to characterize both the metabolites and proteins present under standard laboratory growth conditions. We have assessed the extracellular milieu of Nostoc punctiforme when grown in liquid culture in the presence and absence of a nitrogen source (nitrate). The extracellular proteins identified were enriched in integrin β-propellor domains and calcium-binding sites with sequences unique to N. punctiforme, supporting a role for extracellular proteins in modulating species-specific recognition and behavior processes. Extracellular proteases are present and active under both conditions, with the cells grown with nitrate having a higher activity when normalized to chlorophyll levels. The released metabolites are enriched in peptidoglycan-derived tetrasaccharides, with higher levels in nitrate-free media.

Infections with the vascular pathogens Verticillium longisporum and Verticillium dahliae induce distinct disease symptoms and differentially affect drought stress tolerance of Arabidopsis thaliana

Verticillium longisporum and Verticillium dahliae are soil borne vascular pathogens. Under standard laboratory growth conditions both fungi infect Arabidopsis thaliana ecotype Col-0 plants via primary and lateral (secondary) root tips and colonize the vascular cylinder including proto- and metaxylem. After entry into the hypocotyl both Verticillium species spread into petioles and leaves, but with different efficiency and distinct disease symptom patterns. V. longisporum typically induces early senescence whereas V. dahliae infections cause wilting. In both cases, these symptoms coincide with the switch from a biotrophic to a necrotrophic life style. Fungal proliferation analyses monitored by quantitative real-time PCR and anatomical studies show that V. dahliae is able to colonize aerial plant parts more efficiently compared to V. longisporum. Moreover, V. longisporum-infection triggers hyperplastic xylem formation and cellular transdifferentiation in Arabidopsis, whereas V. dahliae-infection induces enhanced xylem lignification. We have recently shown that V. longisporum induced de novo xylem formation results in enhanced drought stress tolerance, suggesting a conditionally mutualistic interaction. Here we show that on the contrary V. dahliae infections do not provide protection of the plant against drought. Together, our results demonstrate that V. dahliae and V. longisporum induce distinct and species-specific developmental and structural alterations in Arabidopsis, which differently affect plant fitness under concomitant abiotic drought stress conditions.

Transmission of an Oxygen Availability Signal at the Salmonella enterica Serovar Typhimurium fis Promoter

The nucleoid-associated protein FIS is a global regulator of gene expression and chromosome structure in Escherichia coli and Salmonella enterica. Despite the importance of FIS for infection and intracellular invasion, very little is known about the regulation of S. enterica fis expression. Under standard laboratory growth conditions, fis is highly expressed during rapid growth but is then silenced as growth slows. However, if cells are cultured in non-aerated conditions, fis expression is sustained during stationary phase. This led us to test whether the redox-sensing transcription factors ArcA and FNR regulate S. enterica fis. Deletion of FNR had no detectable effect, whereas deletion of ArcA had the unexpected effect of further elevating fis expression in stationary phase. ArcA required RpoS for induction of fis expression, suggesting that ArcA indirectly affects fis expression. Other putative regulators were found to play diverse roles: FIS acted directly as an auto-repressor (as expected), whereas CRP had little direct effect on fis expression. Deleting regions of the fis promoter led to the discovery of a novel anaerobically-induced transcription start site (Pfis-2) upstream of the primary transcription start site (Pfis-1). Promoter truncation also revealed that the shortest functional fis promoter was incapable of sustained expression. Moreover, fis expression was observed to correlate directly with DNA supercoiling in non-aerated conditions. Thus, the full-length S. enterica fis promoter region may act as a topological switch that is sensitive to stress-induced duplex destabilisation and up-regulates expression in non-aerated conditions.

Mapping Yeast Transcriptional Networks

The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.

Impairment of O-antigen production confers resistance to grazing in a model amoeba–cyanobacterium predator–prey system

The grazing activity of predators on photosynthetic organisms is a major mechanism of mortality and population restructuring in natural environments. Grazing is also one of the primary difficulties in growing cyanobacteria and other microalgae in large, open ponds for the production of biofuels, as contaminants destroy valuable biomass and prevent stable, continuous production of biofuel crops. To address this problem, we have isolated a heterolobosean amoeba, HGG1, that grazes upon unicellular and filamentous freshwater cyanobacterial species. We have established a model predator-prey system using this amoeba and Synechococcus elongatus PCC 7942. Application of amoebae to a library of mutants of S. elongatus led to the identification of a grazer-resistant knockout mutant of the wzm ABC O-antigen transporter gene, SynPCC7942_1126. Mutations in three other genes involved in O-antigen synthesis and transport also prevented the expression of O-antigen and conferred resistance to HGG1. Complementation of these rough mutants returned O-antigen expression and susceptibility to amoebae. Rough mutants are easily identifiable by appearance, are capable of autoflocculation, and do not display growth defects under standard laboratory growth conditions, all of which are desired traits for a biofuel production strain. Thus, preventing the production of O-antigen is a pathway for producing resistance to grazing by certain amoebae.

The Interplay between Protein L-Isoaspartyl Methyltransferase Activity and Insulin-Like Signaling to Extend Lifespan in Caenorhabditis elegans

The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS) pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair.

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961.

A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein.

Clostridium difficile is a nosocomial pathogen that can cause severe gastrointestinal infections. C. difficile encodes a family of cell wall proteins, some of which are implicated in pathogenesis. Here we have characterized CwpV, the largest member of this family. CwpV is surface expressed and post-translationally processed in a manner analogous to the major S-layer protein SlpA. Expression of cwpV is phase variable, with approximately 5% of cells in a population expressing the protein under standard laboratory growth conditions. Upstream of cwpV, inverted repeats flank a 195 bp sequence which undergoes DNA inversion. Use of a gusA transcriptional reporter demonstrated that phase variation is mediated by DNA inversion; in one orientation cwpV is expressed while in the opposite orientation the gene is silent. The inversion region contains neither the promoter nor any of the open reading frame, therefore this system differs from previously described phase variation mechanisms. The cwpV promoter is located upstream of the inversion region and we propose a model of phase variation based on intrinsic terminator formation in the OFF transcript. A C. difficile site-specific recombinase able to catalyse the inversion has been identified.

Mixed fermentation for natural product drug discovery

Natural products continue to play a major role in drug discovery and development. However, chemical redundancy is an ongoing problem. Genomic studies indicate that certain groups of bacteria and fungi have dozens of secondary metabolite pathways that are not expressed under standard laboratory growth conditions. One approach to more fully access the metabolic potential of cultivatable microbes is mixed fermentation, where the presence of neighboring microbes may induce secondary metabolite synthesis. Research to date indicates that mixed fermentation can result in increased antibiotic activity in crude extracts, increased yields of previously described metabolites, increased yields of previously undetected metabolites, analogues of known metabolites resulting from combined pathways and, importantly, induction of previously unexpressed pathways for bioactive constituents.

Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

Normal expression and function of transfer RNA (tRNA) are of paramount importance for translation. In this study, we show that tRNA defects are also associated with increased levels of immature ribosomal RNA (rRNA). This association was first shown in detail for a mutant strain that underproduces tRNA(Arg2) in which unprocessed 16S and 23S rRNA levels were increased several-fold. Ribosome profiles indicated that unprocessed 23S rRNA in the mutant strain accumulates in ribosomal fractions that sediment with altered mobility. Underproduction of tRNA(Arg2) also resulted in growth defects under standard laboratory growth conditions. Interestingly, the growth and rRNA processing defects were attenuated when cells were grown in minimal medium or at low temperatures, indicating that the requirement for tRNA(Arg2) may be reduced under conditions of slower growth. Other tRNA defects were also studied, including a defect in RNase P, an enzyme involved in tRNA processing; a mutation in tRNA(Trp) that results in its degradation at elevated temperatures; and the titration of the tRNA that recognizes rare AGA codons. In all cases, the levels of unprocessed 16S and 23S rRNA were enhanced. Thus, a range of tRNA defects can indirectly influence translation via effects on the biogenesis of the translation apparatus.

Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae

The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.

Identification of Mycobacterium avium Genes That Affect Invasion of the Intestinal Epithelium

Invasion of intestinal mucosa of the host by Mycobacterium avium is a critical step in pathogenesis and likely involves several different bacterial proteins, lipids, glycoproteins, and/or glycolipids. Through the screening of an M. avium genomic library in Mycobacterium smegmatis, we have identified a number of M. avium genes that are associated with increased invasion of mucosal epithelial cells. In order to further investigate these genes, we cloned six of them into a plasmid downstream of a strong mycobacterial promoter (L5 mycobacterial phage promoter), resulting in constitutive expression. Bacteria were then evaluated for increased expression and examined for invasion of HT-29 intestinal epithelial cells. The genes identified encode proteins that are similar to (i) M. tuberculosis coenzyme A carboxylase, (ii) M. tuberculosis membrane proteins of unknown function, (iii) M. tuberculosis FadE20, (iv) a Mycobacterium paratuberculosis surface protein, and (v) M. tuberculosis cyclopropane fatty acyl-phopholipid synthase. The constitutive expression of these genes confers to M. avium the ability to invade HT-29 intestinal epithelial cells with a severalfold increase in efficiency compared to both the wild-type M. avium and M. avium containing the vector alone. Using the murine intestinal ligated loop model, it was observed that the constitutive expression of M. avium proteins has a modest impact on the ability to enter the intestinal mucosa when compared with the wild-type control, suggesting that under in vivo conditions these genes are expressed at higher levels. Evaluation of the expression of these invasion-related genes indicated that under conditions similar to the intestinal lumen environment, the genes identified are upregulated. These data suggest that invasion of the intestinal mucosa is an event that requires the participation of several bacterial factors and the expression of the genes that encode them is less observed under standard laboratory growth conditions.

Identification of a gene that affects the efficiency of host cell infection by Legionella pneumophila in a temperature-dependent fashion.

The ability to infect host cells is critical for the survival and replication of intracellular pathogens in humans. We previously found that many genes involved in the ability of Legionella pneumophila to infect macrophages are not expressed efficiently under standard laboratory growth conditions. We have developed an approach using expression of L. pneumophila genes from an exogenous constitutive promoter on a low-copy-number vector that allows identification of genes involved in host cell infection. Through the use of this strategy, we found that expression of a gene, lvhB2, enhances the efficiency of L. pneumophila infection of mammalian cells. The putative protein encoded by lvhB2 has similarity to structural pilin subunits of type IV secretion systems. We confirmed that this gene plays a role in host cell infection by the construction of an in-frame deletion in the L. pneumophila lvhB2 gene and complementation of this mutant with the wild-type gene. The lvhB2 mutant does not display a very obvious defect in interactions with host cells when the bacteria are grown at 37 degrees C, but it has an approximately 100-fold effect on entry and intracellular replication when grown at 30 degrees C. These data suggest that lvhB2 plays an important role in the efficiency of host cell infection by L. pneumophila grown at lower temperatures.

Genome engineering reveals large dispensable regions in Bacillus subtilis.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.