Three Microbial Musketeers of the Seas: Shewanella baltica, Aliivibrio fischeri and Vibrio harveyi, and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.

Bacteriophage-encoded enzymes destroying bacterial cell membranes and walls, and their potential use as antimicrobial agents.

Appearance of pathogenic bacteria resistant to most, if not all, known antibiotics is currently one of the most significant medical problems. Therefore, development of novel antibacterial therapies is crucial for efficient treatment of bacterial infections in the near future. One possible option is to employ enzymes, encoded by bacteriophages, which cause destruction of bacterial cell membranes and walls. Bacteriophages use such enzymes to destroy bacterial host cells at the final stage of their lytic development, in order to ensure effective liberation of progeny virions. Nevertheless, to use such bacteriophage-encoded proteins in medicine and/or biotechnology, it is crucial to understand details of their biological functions and biochemical properties. Therefore, in this review article, we will present and discuss our current knowledge on the processes of bacteriophage-mediated bacterial cell lysis, with special emphasis on enzymes involved in them. Regulation of timing of the lysis is also discussed. Finally, possibilities of the practical use of these enzymes as antibacterial agents will be underlined and perspectives of this aspect will be presented.

The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli.

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates.

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

Various modes of action of dietary phytochemicals, sulforaphane and phenethyl isothiocyanate, on pathogenic bacteria.

Isothiocyanates (ITCs) derived from cruciferous plants reveal antibacterial activity, although detailed mechanism is not fully elucidated. Recently it has been reported that ITCs induce the stringent response in Escherichia coli strains. The aim of this work was to determine whether two isothiocyanates, sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), similarly as in E. coli induce stringent response in Bacillus subtilis, model Gram(+) bacterium, and test their potency against a panel of clinical isolates belonging to Gram(+) or Gram(-) groups. Minimal inhibitory concentrations were determined as well as effect of ITCs on membranes integrity, synthesis of DNA, RNA and stringent response alarmones was assessed. SFN and PEITC are effective against B. subtilis and bacterial isolates, namely E. coli, K. pneumonia, S. aureus, S. epidermidis and E. faecalis. Interestingly, in B. subtilis and E. faecalis the inhibition of growth and nucleic acids synthesis is independent of ppGpp accumulation. In bacteria, which do not induce the stringent response in the presence of ITCs, membrane integrity disruption is observed. Thus, ITCs are effective against different pathogenic bacteria and act by at least two mechanisms depending on bacteria species.

Multiple links connect central carbon metabolism to DNA replication initiation and elongation in Bacillus subtilis.

DNA replication is coupled to growth by an unknown mechanism. Here, we investigated this coupling by analyzing growth and replication in 15 mutants of central carbon metabolism (CCM) cultivated in three rich media. In about one-fourth of the condition tested, defects in replication resulting from changes in initiation or elongation were detected. This uncovered 11 CCM genes important for replication and showed that some of these genes have an effect in one, two or three media. Additional results presented here and elsewhere (Jannière, L., Canceill, D., Suski, C., et al. (2007), PLoS One, 2, e447.) showed that, in the LB medium, the CCM genes important for DNA elongation (gapA and ackA) are genetically linked to the lagging strand polymerase DnaE while those important for initiation (pgk and pykA) are genetically linked to the replication enzymes DnaC (helicase), DnaG (primase) and DnaE. Our work thus shows that the coupling between growth and replication involves multiple, medium-dependent links between CCM and replication. They also suggest that changes in CCM may affect initiation by altering the functional recruitment of DnaC, DnaG and DnaE at the chromosomal origin, and may affect elongation by altering the activity of DnaE at the replication fork. The underlying mechanism is discussed.

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.

Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus.

Phenethyl isothiocyanate inhibits shiga toxin production in enterohemorrhagic Escherichia coli by stringent response induction.

The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) depends on production of Shiga toxins, which are encoded by stx genes located in the genomes of lambdoid prophages. Efficient expression of these genes requires prophage induction and lytic development of phages. Treatment of EHEC infections is problematic due to not only the resistance of various strains to antibiotics but also the fact that many antibiotics cause prophage induction, thus resulting in high-level expression of stx genes. Here we report that E. coli growth, Shiga toxin-converting phage development, and production of the toxin by EHEC are strongly inhibited by phenethyl isothiocyanate (PEITC). We demonstrate that PEITC induces the stringent response in E. coli that is mediated by massive production of a global regulator, guanosine tetraphosphate (ppGpp). The stringent response induction arises most probably from interactions of PEITC with amino acids and from amino acid deprivation-mediated activation of ppGpp synthesis. In mutants unable to synthesize ppGpp, development of Shiga toxin-converting phages and production of Shiga toxin are significantly enhanced. Therefore, ppGpp, which appears at high levels in bacterial cells after stimulation of its production by PEITC, is a negative regulator of EHEC virulence and at the same time efficiently inhibits bacterial growth. This is in contrast to stimulation of virulence of different bacteria by this nucleotide reported previously by others.

Different effects of ppGpp on Escherichia coli DNA replication in vivo and in vitro.

Inhibition of Escherichia coli DNA replication by guanosine tetraphosphate (ppGpp) is demonstrated in vitro. This finding is compatible with impairment of the DnaG primase activity by this nucleotide. However, in agreement to previous reports, we were not able to detect a rapid inhibition of DNA synthesis in E. coli cells under the stringent control conditions, when intracellular ppGpp levels increase dramatically. We suggest that the process of ppGpp-mediated inhibition of DnaG activity may be masked in E. coli cells, which could provide a rationale for explanation of differences between ppGpp effects on DNA replication in E. coli and Bacillus subtilis.

Replicating DNA by cell factories: roles of central carbon metabolism and transcription in the control of DNA replication in microbes, and implications for understanding this process in human cells.

Precise regulation of DNA replication is necessary to ensure the inheritance of genetic features by daughter cells after each cell division. Therefore, determining how the regulatory processes operate to control DNA replication is crucial to our understanding and application to biotechnological processes. Contrary to early concepts of DNA replication, it appears that this process is operated by large, stationary nucleoprotein complexes, called replication factories, rather than by single enzymes trafficking along template molecules. Recent discoveries indicated that in bacterial cells two processes, central carbon metabolism (CCM) and transcription, significantly and specifically influence the control of DNA replication of various replicons. The impact of these discoveries on our understanding of the regulation of DNA synthesis is discussed in this review. It appears that CCM may influence DNA replication by either action of specific metabolites or moonlighting activities of some enzymes involved in this metabolic pathway. The role of transcription in the control of DNA replication may arise from either topological changes in nucleic acids which accompany RNA synthesis or direct interactions between replication and transcription machineries. Due to intriguing similarities between some prokaryotic and eukaryotic regulatory systems, possible implications of studies on regulation of microbial DNA replication on understanding such a process occurring in human cells are discussed.

Mutations in central carbon metabolism genes suppress defects in nucleoid position and cell division of replication mutants in Escherichia coli.

A genetic link of the carbon metabolism and DNA replication was recently reported for the representative of Gram-negative bacteria, Escherichia coli. Our studies showed that the viability of thermosensitive replication mutants at high temperature can be improved or fully recovered by deleting certain genes of central carbon metabolism (CCM). In order to improve our understanding of this phenomenon, in this study we analyzed the length and nucleoid distribution of suppressed thermosensitive replication mutants. The dysfunctions in the replication machinery generally lead to formation of elongated cells (termed filaments) that originate from an inhibition of cell division dependent on replication-stress, and to abnormal distribution and compaction of nucleoids. The results reported here provide evidence that deletion of the pta and ackA CCM genes significantly reduces observed cell length in the replication mutants dnaA46, dnaB8, dnaE486, dnaG(ts) and dnaN159. A weaker effect was shown in the tktB dnaE486 double mutant. The CCM enzyme dysfunction restored also the nucleoid shape and position in double mutants. The specificity of these effects was confirmed by overexpression of fully functional genes coding for relevant CCM enzymes, which caused the reversion to the initial filamentous and nucleoid phenotypes. These results indicate that CCM mutations can rescue (or reduce) the cell division defects resulting from various replication mutations. We thus suggest that the replication-metabolism connection may serve as a general mechanism affecting DNA duplication at various levels to adjust this process and the cell division to the status of cell physiology.