- Volume 159, Issue Pt_9, 2013

The outer membrane lipoprotein RcsF is an essential component of the Rcs phosphorelay signal transduction system in Escherichia coli. It senses stresses imposed on the cell envelope and conveys the information to histidine kinase RcsC in the cytoplasmic membrane. Mislocalization of RcsF to the periplasm, effected by fusing it to the periplasmic maltose-binding protein, or to the cytoplasmic membrane, brought about by changing the lipoprotein sorting signal, leads to high activation of the Rcs system, suggesting that RcsF functions as a ligand for RcsC in activating the system. Here, we focus on the proline-rich region (PRR) in the N-terminal half of RcsF, a region which also contains many basic amino acid residues. Deletion of the PRR in the mislocalized RcsF resulted in even higher activation of the Rcs system. The same deletion in wild-type RcsF lipoprotein that is correctly localized to the outer membrane, however, blocked activation of the system under stresses that normally should activate it. It is highly likely that the PRR plays an important role in the regulation of the function of RcsF in activating the Rcs system.

Twitching motility in Acinetobacter baylyi ADP1 is inhibited by moderate intensities of blue light in a temperature-dependent manner (maximally at 20 °C). We analysed the involvement of four predicted blue-light sensing using flavin (BLUF)-domain-containing proteins encoded in the genome of this strain in the twitching motility phenotype. All four genes were expressed both in light and in darkness. A phylogenetic tree showed that one BLUF domain, ACIAD2110, grouped separately from the other three (ACIAD1499, ACIAD2125 and ACIAD2129). Individual knockout mutants of the latter three, but not of ACIAD2110, fully abolished the light dependency of the twitching motility response. Quantitative analysis of transcript level of the three genes showed a decreased expression in the light, with dark/light ratios of 1.65±0.28, 1.79±0.21 and 2.69±0.39, for ACIAD2125, ACIAD2129 and ACIAD1499, respectively. Double and triple knockouts of ACIAD1499, ACIAD2125 and ACIAD2129 confirmed the same phenotype as the corresponding single knockouts. Complementation of all the single knockouts and the triple knockout mutants with any of the three BLUF-domain-encoding genes fully restored the inhibition of twitching motility by blue light that is observed in the wild-type strain. A. baylyi ADP1 therefore shows a high degree of redundancy in the genes that encode BLUF-containing photoreceptors. Moreover, all plasmid-complemented strains, expressing any of the BLUF proteins irrespective of the specific set of deleted photoreceptors, displayed increased light-dependent inhibition of twitching motility, as compared to the wild-type (P<0.001). We conclude that the three genes ACIAD1499, ACIAD2125 and ACIAD2129 are jointly required to inhibit twitching motility under moderate blue-light illumination.

Escherichia coli has five genes encoding l,d-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to cross-link Braun’s lipoprotein to the peptidoglycan (PG), while the other two (YnhG, YcbB) form direct meso-diaminopimelate (DAP-DAP, or 3-3) cross-links within the PG. In addition, Ldt enzymes can also incorporate non-canonical d-amino acids, such as d-methionine, into the PG. To further investigate the role of these enzymes and, in particular, 3-3 linkages in cell envelope physiology we constructed and phenotypically characterized a variety of multiple Ldt deletion mutants of E. coli. We report that a triple deletion mutant lacking ybiS, erfK and ycfS is hypersusceptible to the metal-chelating agent EDTA, leaks periplasmic proteins and is resistant to the toxic effect of d-methionine. A double ynhG ycbB mutant had no discernible phenotype; however, examination of the phenotypes of various Ldt mutants bearing an additional DAP auxotrophic mutation (dapA : : Cm) showed that a quintuple mutant strain lacking all Ldt genes was severely impaired for growth on media with limited DAP. These data demonstrate that loss of the E. coli Ldt enzymes involved with coupling the PG to Braun's lipoprotein resulted in the loss of outer membrane stability while loss of the Ldt enzymes involved with DAP-DAP linkages had no observable effect on the cell envelope. Loss of all Ldt enzymes proved detrimental to growth when cells were starved for DAP, indicating a combined role for both 3-3 and Braun’s lipoprotein cross-links in cell viability only under a specific PG stress.

Sporulation is an essential part of the life cycle of the industrially important filamentous fungus Aspergillus niger. The formation of conidiophores, spore-bearing structures, requires remodelling of the fungal cell wall, as demonstrated by the differences in carbohydrate composition of cell walls of vegetative mycelium and spores. Glycoside hydrolases that are involved in this process have so far remained unidentified. Using transcriptome analysis, we have identified genes encoding putative cell-wall-modifying proteins with enhanced expression in sporulating aerial mycelium compared to vegetative mycelium. Among the most strongly induced genes were those encoding a protein consisting of a putative chitin binding module (CBM14) and the chitinolytic enzymes NagA, CfcI and CtcB. Reporter studies showed that the N-acetyl-β-hexosaminidase gene nagA was expressed both in vegetative hyphae and in aerial structures (aerial hyphae, conidiophores and conidia) upon starvation. In contrast, promoter activities of the chitinase genes ctcB and cfcI were specifically localized in the conidiophores and conidia. CtcB is an endo-chitinase and CfcI releases monomers from chitin oligosaccharides: together these enzymes have the potential to degrade chitin of the fungal cell wall. Inactivation of both the cfcI and ctcB genes affected neither radial growth rate, nor formation and germination of spores. The amount of chitin in the spore walls of a ΔcfcIΔctcB double deletion strain, however, was significantly increased compared with the wild-type, thus indicating that CfcI and CtcB indeed modify the A. niger cell walls during sporulation. These novel insights in the sporulation process in aspergilli are of strong scientific relevance, and also may aid industrial strain engineering.

Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with d-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA–D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers d-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for d-alanine incorporation through a process that has been proposed to proceed via a d-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of d-alanine, indicating that LTA has a role, either direct or indirect, in the efficient d-alanine incorporation into WTA in living cells.

Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotide-mediated transformation of Sulfolobus and other micro-organisms.

Recently, we described a novel insertion element, ISPpy1, isolated from a permafrost strain of Psychrobacter maritimus. In this work, we demonstrated that ISPpy1 is a member of a novel subgroup of the IS3 family of insertion sequences (ISs) that was not identified and characterized previously. IS elements of this subgroup termed the ISPpy1 subgroup are broadly distributed among different taxa of Eubacteria, including Geobacteraceae, Chlorobiaceae, Desulfobacteraceae, Methylobacteriaceae, Nitrosomonadaceae and Cyanobacteria. While displaying characteristic features of the IS3-family elements, ISPpy1 subgroup elements exhibit some unusual features. In particular, most of them have longer terminal repeats with unconventional ends and frameshifting box with an atypical organization, and, unlike many other IS3-family elements, do not exhibit any distinct IS specificity. We studied the transposition and mutagenic properties of a representative member of this subgroup, ISPpy1 and showed that in contrast to the original P. maritimus host, in a heterologous host, Escherichia coli K-12, it is able to translocate with extremely high efficiency into the chromosome, either by itself or as a part of a composite transposon containing two ISPpy1 copies. The majority of transposants carry multiple chromosomal copies (up to 12) of ISPpy1. It was discovered that ISPpy1 is characterized by a marked mutagenic activity in E. coli: its chromosomal insertions generate various types of mutations, including auxotrophic, pleiotropic and rifampicin-resistance mutations. The distribution of IS elements of the novel subgroup among different bacteria, their role in the formation of composite transposons and the horizontal transfer of genes are examined and discussed.

A new generation of vaccines containing multiple protein components that aim to provide broad protection against serogroup B meningococci has been developed. One candidate, 4CMenB (4 Component MenB), has been approved by the European Medicines Agency, but is predicted to provide at most 70–80 % strain coverage; hence there is a need for second-generation vaccines that achieve higher levels of coverage. Prior knowledge of the diversity of potential protein vaccine components is a key step in vaccine design. A number of iron import systems have been targeted in meningococcal vaccine development, including the HmbR and HpuAB outer-membrane proteins, which mediate the utilization of haemoglobin or haemoglobin–haptoglobin complexes as iron sources. While the genetic diversity of HmbR has been described, little is known of the diversity of HpuAB. Using whole genome sequences deposited in a Bacterial Isolate Genome Sequence Database (BIGSDB), the prevalence and diversity of HpuAB among Neisseria were investigated. HpuAB was widely present in a range of Neisseria species whereas HmbR was mainly limited to the pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae. Patterns of sequence variation in sequences from HpuAB proteins were suggestive of recombination and diversifying selection consistent with strong immune selection. HpuAB was subject to repeat-mediated phase variation in pathogenic Neisseria and the closely related non-pathogenic Neisseria species Neisseria lactamica and Neisseria polysaccharea but not in the majority of other commensal Neisseria species. These findings are consistent with HpuAB being subject to frequent genetic transfer potentially limiting the efficacy of this receptor as a vaccine candidate.

Bacteria have evolved a set of regulatory pathways to adapt to the dynamic nutritional environment during the course of infection. However, the underlying mechanism of the regulatory effects by nutritional cues on bacterial pathogenesis is unclear. In the present study, we showed that the Pseudomonas aeruginosa catabolite repression control protein regulates the Pseudomonas quinolone signal quorum sensing, which further controls synthesis of virulence factor pyocyanin, biofilm formation and survival during infection models. Our study suggests that deregulation of the catabolite repression by P. aeruginosa might enhance its fitness during cystic fibrosis infections.

The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure–function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida.

Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between Arabidopsis thaliana and Alt. brassicicola is a representative model system, and objective estimation of disease progression is indispensable for accurate functional analyses. Five strains caused black spot symptom progression on Korean cabbage and Ara. thaliana ecotype Col-0. In particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship between mycelial growth in the infected tissues and symptom progression, we have established a reliable quantification method using real-time PCR that employs a primer pair and dual-labelled probe specific to a unigene encoding A. brassicicola SCYTALONE DEHYDRATASE1 (AbSCD1), which is involved in fungal melanin biosynthesis. Plotting the crossing point values from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877 in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8 times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual in planta ramification. Mycelial growth did not always coincide with symptom development. Our quantitative evaluation system is applicable and reliable for the objective estimation of black spot disease severity.

We conducted experiments in order to examine whether the probiotic Lactobacillus casei strain Shirota YIT9029 (LcS) in vitro and in vivo antagonism of Helicobacter pylori and Salmonella, involves inhibition of the swimming motility of these pathogens. We report the irreversible inhibition of the swimming motility of H. pylori strain 1101 and reversible inhibition of Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1344 by compound(s) secreted by LcS. In H. pylori 1101, irreversible inhibition results in the helical cells being progressively replaced by cells with ‘c’-shaped and coccoid morphologies, accompanied by a loss of FlaA and FlaB flagellin expression. In S. Typhimurium SL1344, transient inhibition develops after membrane depolarization and without modification of expression of FliC flagellin. The inhibitory activity of strain LcS against both S. Typhimurium and H. pylori swimming motilities is linked with a small sized, heat-sensitive, and partially trypsin-sensitive, secreted compound(s), and needed the cooperation of the secreted membrane permeabilizing lactic acid metabolite. The inhibition of S. Typhimurium SL1344 swimming motility leads to delayed cell entry into human enterocyte-like Caco-2/TC7 cells and a strong decrease of cell entry into human mucus-secreting HT29-MTX cells.

Carbon metabolic pathways are important to the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, extremely little is known about metabolic regulation in mycobacteria. There is growing evidence for lysine acetylation being a mechanism of regulating bacterial metabolism. Lysine acetylation is a post-translational modification in which an acetyl group is covalently attached to the side chain of a lysine residue. This modification is mediated by acetyltransferases, which add acetyl groups, and deacetylases, which remove the acetyl groups. Here we set out to test whether lysine acetylation and deacetylation impact acetate metabolism in the model mycobacteria Mycobacterium smegmatis, which possesses 25 candidate acetyltransferases and 3 putative lysine deacetylases. Using mutants lacking predicted acetyltransferases and deacetylases we showed that acetate metabolism in M. smegmatis is regulated by reversible acetylation of acetyl-CoA synthetase (Ms-Acs) through the action of a single pair of enzymes: the acetyltransferase Ms-PatA and the sirtuin deacetylase Ms-SrtN. We also confirmed that the role of Ms-PatA in regulating Ms-Acs regulation depends on cAMP binding. We additionally demonstrated a role for Ms-Acs, Ms-PatA and Ms-SrtN in regulating the metabolism of propionate in M. smegmatis. Finally, along with Ms-Acs, we identified a candidate propionyl-CoA synthetase, Ms5404, as acetylated in whole-cell lysates. This work lays the foundation for studying the regulatory circuit of acetylation and deacetylation in the cellular context of mycobacteria.