Tail-Engineered Phage P2 Enables Delivery of Antimicrobials into Multiple Gut Pathogens.

Bacteriophages can be reprogrammed to deliver antimicrobials for therapeutic and biocontrol purposes and are a promising alternative treatment to antimicrobial-resistant bacteria. Here, we developed a bacteriophage P4 cosmid system for the delivery of a Cas9 antimicrobial into clinically relevant human gut pathogens Shigella flexneri and Escherichia coli O157:H7. Our P4 cosmid design produces a high titer of cosmid-transducing units without contamination by a helper phage. Further, we demonstrate that genetic engineering of the phage tail fiber improves the transduction efficiency of cosmid DNA in S. flexneri M90T as well as allows recognition of a nonnative host, E. coli O157:H7. We show that the transducing units with the chimeric tails enhanced the overall Cas9-mediated killing of both pathogens. This study demonstrates the potential of our P4 cas9 cosmid system as a DNA sequence-specific antimicrobial against clinically relevant gut pathogenic bacteria.

DNA double strand break repair in Escherichia coli perturbs cell division and chromosome dynamics.

To prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum.

Chemical Evolution of a Bacterial Proteome.

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L-ß-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

Bacterial genome instability.

Bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile DNA elements. This implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. In this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. We then discuss the consequences of genome instability at the physiological level, where cells have harnessed instability to mediate phase and antigenic variation, and at the evolutionary level, where horizontal gene transfer has played an important role. Indeed, this ability to share DNA sequences has played a major part in the evolution of life on Earth. The evolutionary plasticity of bacterial genomes, coupled with the vast numbers of bacteria on the planet, substantially limits our ability to control disease.

E. coli SbcCD and RecA control chromosomal rearrangement induced by an interrupted palindrome.

Survival and genome stability are critical characteristics of healthy cells. DNA palindromes pose a threat to genome stability and have been shown to participate in a reaction leading to the formation of inverted chromosome duplications centered around themselves. There is considerable interest in the mechanism of this rearrangement given its likely contribution to genome instability in cancer cells. This study shows that formation of large inverted chromosome duplications can be observed in the chromosome of Escherichia coli. They are formed at the site of a 246 bp interrupted DNA palindrome in the absence of the hairpin nuclease SbcCD and the recombination protein RecA. The genetic requirements for this spontaneous rearrangement are consistent with a pathway involving DNA degradation and hairpin formation, as opposed to a cruciform cleavage pathway. Accordingly, the formation of palindrome-dependent hairpin intermediates can be induced by an adjacent DNA double-stand break.

Overexpression of the single-stranded DNA-binding protein (SSB) stabilises CAG*CTG triplet repeats in an orientation dependent manner.

The stability and deletion-size-distribution profiles of leading strand (CAG)(75) and (CTG)(137) trinucleotide repeat arrays inserted in the Escherichia coli chromosome were investigated upon overexpression of the single-stranded DNA-binding protein (SSB) and in mutant strains deficient for the SbcCD (Rad51/Mre11) nuclease. SSB overexpression increases the stability of the (CAG)(75) repeat array and leads to a loss of the bias towards large deletions for the same array. Furthermore, the absence of SbcCD leads to a reduction in the number of large deletions in strains containing the (CTG)(137) repeat array.

SbcCD regulation and localization in Escherichia coli.

The SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. In Escherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates the regulation of the sbcDC operon and the cellular localization of the SbcC and SbcD proteins. Transcription of the sbcDC operon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm.

A disulfide bond-containing alkaline phosphatase triggers a BdbC-dependent secretion stress response in Bacillus subtilis.

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for alpha-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.

The Escherichia coli rpoS-dependent htrC gene is not involved in the heat shock response.

We found that a new mutant with a deletion/replacement of the Escherichia coli K-12 htrC gene, a gene previously reported to be required for growth at elevated temperatures, is not temperature sensitive. Furthermore, the original mutants, kindly provided by the original authors, although temperature sensitive, do not have mutations in the open reading frame designated htrC. We found that htrC requires RpoS for enhanced expression in the early stationary phase and is expressed at very low levels until then. The growth of our htrC mutant slowed during the early stationary phase, and the mutant was replaced by its parent in mixed cultures. Since we cannot assign a function or distinctive phenotype to htrC, we suggest that this open reading frame should be given a positional designation, yjaZ, until a specific function is identified.

The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis.

Bacillus species are valuable producers of industrial enzymes and biopharmaceuticals, because they can secrete large quantities of high-quality proteins directly into the growth medium. This requires the concerted action of quality control factors, such as folding catalysts and 'cleaning proteases'. The expression of two important cleaning proteases, HtrA and HtrB, of Bacillus subtilis is controlled by the CssRS two-component regulatory system. The induced CssRS-dependent expression of htrA and htrB has been defined as a protein secretion stress response, because it can be triggered by high-level production of secreted alpha-amylases. It was not known whether translocation of these alpha-amylases across the membrane is required to trigger a secretion stress response or whether other secretory proteins can also activate this response. These studies show for the first time that the CssRS-dependent response is a general secretion stress response which can be triggered by both homologous and heterologous secretory proteins. As demonstrated by high-level production of a nontranslocated variant of the alpha-amylase, AmyQ, membrane translocation of secretory proteins is required to elicit this general protein secretion stress response. Studies with two other secretory reporter proteins, lipase A of B. subtilis and human interleukin-3, show that the intensity of the protein secretion stress response only partly reflects the production levels of the respective proteins. Importantly, degradation of human interleukin-3 by extracellular proteases has a major impact on the production level, but only a minor effect on the intensity of the secretion stress response.

Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome.

Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

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.

The extracellular proteome of Bacillus subtilis under secretion stress conditions.

The accumulation of malfolded proteins in the cell envelope of the Gram-positive eubacterium Bacillus subtilis was previously shown to provoke a so-called secretion stress response. In the present studies, proteomic approaches were employed to identify changes in the extracellular proteome of B. subtilis in response to secretion stress. The data shows that, irrespective of the way in which secretion stress is imposed on the cells, the levels of only two extracellular proteins, HtrA and YqxI, display major variations in a parallel manner. Whereas the extracellular level of the HtrA protease is determined through transcriptional regulation, the level of YqxI in the growth medium is determined post-transcriptionally in an HtrA-dependent manner. In the absence of secretion stress, the extracellular levels of HtrA and YqxI are low because of extracytoplasmic proteolysis. Finally, the protease active site of HtrA is dispensable for post-transcriptional YqxI regulation. It is known that Escherichia coli HtrA has combined protease and chaperone-like activities. As this protein shares a high degree of similarity with B. subtilis HtrA, it can be hypothesized that both activities are conserved in B. subtilis HtrA. Thus, a chaperone-like activity of B. subtilis HtrA could be involved in the appearance of YqxI on the extracellular proteome.

A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis.

Bacteria need dedicated systems that allow appropriate adaptation to the perpetual changes in their environments. In Bacillus subtilis, two HtrA-like proteases, HtrA and HtrB, play critical roles in the cellular response to secretion and heat stresses. Transcription of these genes is induced by the high-level production of a secreted protein or by a temperature upshift. The CssR-CssS two-component regulatory system plays an essential role in this transcriptional activation. Transcription of the cssRS operon is autoregulated and can be induced by secretion stress, by the absence of either HtrA or HtrB, and by heat stress in a HtrA null mutant strain. Two start sites are used for cssRS transcription, only one of which is responsive to heat and secretion stress. The divergently transcribed htrB and cssRS genes share a regulatory region through which their secretion and heat stress-induced expression is linked. This study shows that CssRS-regulated genes represent a novel class of heat-inducible genes, which is referred to as class V and currently includes two genes: htrA and htrB.