Kenneth E. Rudd

Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli

BACKGROUNDnThe 1.83 Megabase (Mb) sequence of the Haemophilus influenzae chromosome, the first completed genome sequence of a cellular life form, has been recently reported. Approximately 75 % of the 4.7 Mb genome sequence of Escherichia coli is also available. The life styles of the two bacteria are very different - H. influenzae is an obligate parasite that lives in human upper respiratory mucosa and can be cultivated only on rich media, whereas E. coli is a saprophyte that can grow on minimal media. A detailed comparison of the protein products encoded by these two genomes is expected to provide valuable insights into bacterial cell physiology and genome evolution.nnnRESULTSnWe describe the results of computer analysis of the amino-acid sequences of 1703 putative proteins encoded by the complete genome of H. influenzae. We detected sequence similarity to proteins in current databases for 92 % of the H. influenzae protein sequences, and at least a general functional prediction was possible for 83 %. A comparison of the H. influenzae protein sequences with those of 3010 proteins encoded by the sequenced 75 % of the E. coli genome revealed 1128 pairs of apparent orthologs, with an average of 59 % identity. In contrast to the high similarity between orthologs, the genome organization and the functional repertoire of genes in the two bacteria were remarkably different. The smaller genome size of H. influenzae is explained, to a large extent, by a reduction in the number of paralogous genes. There was no long range colinearity between the E. coli and H. influenzae gene orders, but over 70 % of the orthologous genes were found in short conserved strings, only about half of which were operons in E. coli. Superposition of the H. influenzae enzyme repertoire upon the known E. coli metabolic pathways allowed us to reconstruct similar and alternative pathways in H. influenzae and provides an explanation for the known nutritional requirements.nnnCONCLUSIONSnBy comparing proteins encoded by the two bacterial genomes, we have shown that extensive gene shuffling and variation in the extent of gene paralogy are major trends in bacterial evolution; this comparison has also allowed us to deduce crucial aspects of the largely uncharacterized metabolism of H. influenzae.

Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli

SummaryThis work describes an approach towards analyzing the regulatory effects of variation of guanosine 3′,5′-bispyrophosphate (ppGpp) basal levels in Escherichia coli during steady state growth. A series of strains was derived by mutating the spoT gene (which encodes the major cellular ppGppase) so as to obtain systematic increments in ppGpp basal levels. These strains differ genetically at the spoT locus and, in some cases, also at the relA locus because of the severity of spoT mutant alleles. Measurements of ppGpp revealed a ten-fold range of basal levels during growth on minimal medium. The empirical relationship between ppGpp concentration and growth rate is a simple linear inverse correlation. Tandem rrnA ribosomal RNA promoters, present on a multicopy plasmid, are shown to be differentially regulated over this range of basal levels. The upstream P1 promoter activity shows an inverse exponential relation to ppGpp concentration whereas the downstream P2 promoter is only weakly affected. We conclude that there are systematic regulatory consequences associated with small changes in ppGpp basal levels during steady state growth that probably are part of a continuum with more dramatic effects observed during the stringent response to amino acid deprivation.

Sequencing and analysis of bacterial genomes

The complete sequences of two small bacterial genomes have recently become available, and those of several more species should follow within the next two years. Sequence comparisons show that the most bacterial proteins are highly conserved in evolution, allowing predictions to be made about the functions of most products of an uncharacterized genome. Bacterial genomes differ vastly in their gene repertoires. Although genes for components of the translation and transcription machinery, and for molecular chaperones, are typically maintained, many regulatory and metabolic systems are absent in bacteria with small genomes. Mycoplasma genitalium, with the smallest known genome of any cellular life form, lacks virtually all known regulatory genes, and its gene expression may be regulated differently than in other bacteria. Genome organization is evolutionarily labile: extensive gene shuffling leaves only very few conserved gene arrays in distantly related bacteria.

[18] Protein sequence comparison at genome scale

An adequate set of computer procedures tailored to address the task of genome-scale analysis of protein sequences will greatly increase the beneficial impact of the genome sequencing projects on the progress of biological research. This is especially pertinent given the fact that, for model organisms, one-half or more of the putative gene products have not been functionally characterized. Here we described several programs that may comprise the core of such a set and their application to the analysis of about 3000 proteins comprising 75% of the E. coli gene products. We find that the protein sequences encoded in this model genome are a rich source of information, with biologically relevant similarities detected for more than 80% of them. In the majority of cases, these similarities become evident directly from the results of BLAST searches. However, methods for motif analysis provide for a significant increase in search sensitivity and are particularly important for the detection of ancient conserved regions. As a result of sequence similarity analysis, generalized functional predictions can be made for the majority of uncharacterized ORF products, allowing efficient focusing of experimental effort. Clustering of the E. coli proteins on the basis of sequence similarity shows that almost one-half of the bacterial proteins have at least one paralog and that the likelihood that a protein belongs to a small or a large cluster depends on the function of this particular protein.

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF‐binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.

The second aconitase (AcnB) of Escherichia coli

The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kanR mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2 center dot 85 min (131 center dot 6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infection with lambdaacnB phages from the Kohara lambda-E. coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter. The AcnB protein was purified to > or = 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of Mr 100 000 (SDS-PAGE) and 105 000 (gel filtration analysis) compared with Mr 93 500 predicted from the nucleotide sequence. The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).

Immunochemical structure of the OmpD porin from Salmonella typhimurium

The OmpD porin was isolated and purified from Salmonella typhimurium strain SH 7454 (ompC::Tn10), digested with cyanogen bromide (CNBr) and the peptide fragments were separated by SDS-PAGE. N-terminal sequencing identified a total of 96 residues from four distinct peptides. The sequence showed that OmpD is homologous to NmpC (75% identity), Lc(75%) and OmpC (70%) from Escherichia coli, and OmpC (68%) from S. typhimurium. The sequence was essentially identical to the translated sequence of an nmpC-like gene of S. typhimurium, currently placed at 38.6 centisomes of the chromosome. Our results and other data suggest, however, that this gene is actually the ompD gene, which is more correctly placed in the 34 centisome region of the chromosome. The CNBr-generated peptides were also screened with 16 anti-S. typhimurium OmpD monoclonal antibodies by Western blotting. These results, in conjunction with the prediction of the OmpD folding pattern based on the known three-dimensional structure of E. coli OmpF, showed a close immunological relationship among S. typhimurium OmpD and E. coli NmpC and Lc, and a strong conservation of sequences within the transmembrane beta strands of these porins and E. coli OmpC, PhoE and OmpF, and Salmonella typhic OmpC.

Redesigning, implementing and integrating Escherichia coli genome software tools with an object-oriented database system

This paper reports our exploratory work to redesign, implement and integrate a collection of genome software tools with an object-oriented database system. Our software tools deal with genome data from Escherichia coli K-12, a bacterium that has been studied intensively and provides richer data sets than any other living organism. The object-oriented DBMS used for the integration is ONTOS, a commercial object-oriented system from Ontologic Inc. This redesign and implementation task was performed in two steps. First, C programs were converted into C++, and then the C++ version programs were modified and integrated with an object-oriented modeling of the data to form an ONTOS database application. The first step helps us develop a conceptual view for a DBMS-independent object-oriented construct. The second step elucidates what additional DBMS-dependent modification steps are needed to provide persistency to the objects. Examples are included to illustrate steps of the redesign and implementation. Overall, the outcome of this project demonstrates that programs and data can be successfully integrated with an object-oriented database, while providing the objects with persistency and shareability. This paper includes discussions using concrete examples on what advantage the object-oriented database approach provides over the relational database approach.

Escherichia Coli — Functional and Evolutionary Implications of Genome Scale Computer-Aided Protein Sequence Analysis

Complete sequencing of model genomes has recently become a reality. Hundreds of viral and more than 20 organellar genome sequences are currently available (Bork et al., 1994). The genomes of several bacteria, Archaea, and yeast are expected to be completed within 2–3 years. The ultimate value of genome projects is not to establish complete and accurate nucleotide sequences per se, but rather to use the sequence in order to deduce how the genome determines all cellular functions. Eventually, it should be possible to determine the whole pathway from the nucleotide sequence to the phenotype of an organism, which could be re-stated as the “first principles” of cellular structure and function. Numerous biochemical and genetic experiments will be indispensable for achieving this ambitious goal. Nonetheless, computer analysis of the amino acid sequences encoded in the genome is a necessary and complementary approach that allows one to systematically predict protein functions and derive possible evolutionary relationships. One cannot help but to note that computer-assisted sequences analysis, even though generally lacking the precision that is, at least in principle, achievable in laboratory experiments, is much less labor-consuming and costly. In fact, at this time, only computer methods allow one to analyze gene products encoded in a complete genome simultaneously and consistently and to obtain meaningful, readily comparable results for each of them in a relatively short time.